Yazarlar Lynne Spencer NMR spektroskopisi kısmı deney sırasında ona destek için teşekkür etmek istiyorum. Bu çalışma, Ulusal Sağlık fon R01 HL059246 Enstitüleri, R01 HL067970, R01 HL088634 (Dr Tian) ve HL096284 F32 (Dr Kolwicz) hibe ile desteklendi.

<ol><li> Bu deneyler için, aynı anda iki ayrı sistem kullanılmaktadır. 31 P spektrumları edinimi için Bruker 14T mıknatıs Avance III konsol ve topspin V2.1 yazılımı ile donatılmış bir bilgisayar arayüzü. Kalp fonksiyonunun değerlendirilmesi için, özel bir kalp perfüzyon sistemi, veri analizi için LabChartPro 6 yazılımı ile donatılmış, PowerLab 4 / 30 veri toplama ile arabirim. </li><li> 0.5mm EDTA, 5.3mM KCl, 1.2mm MgSO 4, 118mm NaCl, ve 25mm NaHCO 3: Deney günü, Krebs-Henseleit tampon 1 litre şu şekilde hazırlanır. Bu karışım daha sonra% 5 CO 2 / O 2 2mm CaCl 2 ek olarak 10-15 dakika önce için% 95 ile kabarmış . Son olarak, yüzeylerde, 10 mM glukoz ve 0.5mm piruvat şeklinde eklenir. </li><li> Deney sırasında sıcaklık düzenleme kritik öneme sahiptir. Isıtmalı sirkülatörler 37.0 arasındaki sıcaklık korumak için kullanılır - 37.5 ° C kalp mıknatıs içinde iken. Sıcaklık, bir fiber optik sıcaklık probu kullanarak deney boyunca izlenir. </li><li> Perfüzyon basıncı ve sol ventrikül basıncı basınç dönüştürücüleri bir veri toplama sistemi ve verilen yazılım kullanılarak görüntülenir üzerinden takip edilmektedir. Bu deneyde önce standart bir tansiyon aleti ile kalibre edilir. Buna ek olarak, basınç hatları tüm hava kabarcıklarını çıkarmak için yeterince kızardı. </li><li> 150 mM sodyum fosfat (KH tamponun iyonik gücüne eşdeğer olduğu) standart bir örnek, önce kalp ekleme prob &quot;kalibre&quot; için kullanılır. Bu sinyal kolaylaştırır ve kalp prob içinde konumlandırılmış bir kez satın dönemi başlamak için gereken süreyi azaltır. </li><li> Koagülasyon azaltmak için, fare, 5 dakika sonra heparin IP 200 Üniteleri, sodyum pentobarbital (175 mg / kg) IP verilir enjekte edilir. </li><li> Kalp hızla eksize (sağlam akciğerler ve timus ile) ve buz KH tampon tutuklandı. </li><li> Buz üzerinde tutarken, akciğerleri hızla kaldırılır. Timus lobları tanımlanır ve aort yavaşça ortaya çıkarmak için geri soyulmuş. Timus kaldırılır. Aort sonra herhangi bir çevre doku dikkatlice kaldırarak izole edilmiştir. </li><li> Mikro dikiş forseps yavaşça aort hem de duvarları lümen maruz tutmak için kullanılır. Aort 0,965 mm OD polietilen boru (PE50) yapılan kanül dikkatlice yerleştirilir. Sütür aort etrafında hızla bağlı aort mikro damar kıskacı ile yerinde tutulur. Klip kaldırılır ve forseps dikkatle kanül aort kökü üzerinde olduğunu kontrol etmek için kullanılır. Ek bağları kalp yerinde tutmak için gerekli olarak eklenir. </li><li> Herhangi bir ekstra doku forseps ve microscissors kullanarak kaldırılır. Sol kulak kepçesi içine küçük bir kesi yapılır. Yavaşça kalp tutarken A 0.61 mm OD polietilen boru (PI10), apeks ile sol atriyum, sol ventrikül kavite ve dışarı ile dikkatlice eklenir. Aşırı boru kesilir. </li><li> Deflate bir su dolu balon, AG içine atrium sokulur ve yapışkan bant veya sütür kullanılarak yerinde tutulur. Peristaltik pompa hızı yavaş yavaş kalp için yeterli akışını sağlamak için artar. Kalp NMR prob içine yerine kadar, yaklaşık 2 ml / dk eşdeğer sürekli akışı ile kalp perfüze olmaya devam edecektir. LV basınç dönüştürücü çalışıp çalışmadığını doğrulamak için bir mikrometre şırınga kullanarak küçük bir hacmi ile AG balon şişirilir. </li><li> Kalp dikkatlice 10 mm NMR tüpe eklenir. Geniş bir delik &quot;spinner&quot;, prob içinde uygun pozisyona tüp yardımcı olmak için kullanılır. Tüm aparatları sonra yapışkan bant ile sıkıca &quot;göbek kordonu&quot; eklenir. </li><li> Kalp / NMR tüpü, 10 mm NMR prob bobin içinde kadar göbek kordonu mıknatıs üst delik içine yavaş yavaş indirilir. </li><li> Kalp prob içinde uygun pozisyonda sonra, peristaltik pompa akış 80mmHg bir perfüzyon basıncı elde etmek için ayarlanır. (Bu noktaya kadar sürekli bir akış ile yaklaşık 2 ml / dak kalp perfüze olduğunu unutmayın). Perfüzyon basıncı sonra pompa kontrol &quot;hold&quot; mekanizması sağlayarak devam eder. Kalp, 15-20 dakika sonra bir dengeleme dönemi izin verilir. Bu süre zarfında, LV balonun hacmi 8-10 mmHg diyastol sonu basıncı elde etmek için ayarlanır. </li><li> Dengeleme dönemde, mümkün olan en iyi fosfor sinyal elde etmek için spektrometre parametrelerini optimize etmek için gereklidir. Bu radyo darbe ayarı ve fosfor çekirdeği rezonansa hangi frekansta (&quot;ayar&quot;) (&quot;layneri&quot;) manyetik alan homojen hale tarafından gerçekleştirilir. </li><li> Dengelenme periyodundan sonra, birden fazla 31 P NMR spektrumları elde edilebilir. Her spektrum için satın alma süresi t bağlıdır.alan mıknatısın gücü, örnek boyutu ve sinyal gürültü oranı belirli bir deney için gerekli. Spectra, 2.0 saniyede bir 60 derece çevirme açısı ve gecikmeler ile 20 ms&#39;den 256 radyo frekans darbeleri elde sinyal ortalama 14 Telsa mıknatıs kullanarak elde edilir. Bu deney, yaklaşık 10 dakika gerekecektir. </li></ol> Temsilcisi Sonuçlar Veri toplama ve donanım, LabChart yazılım, kalp fonksiyonu çeşitli parametreler deney protokolünde boyunca ölçülebilir. Kalp fonksiyonu tipik ölçüsü, sol ventrikül gelişmiş basıncı (LVDevP), (Şekil 1) sistolik basınç, diyastol sonu basıncı (EDP) çıkarılarak elde edilir. Bu tedbir, fare zorlanma ve kalp (yani, basınç yükü) durumuna bağlı olarak değişebilir. Ancak, tipik olarak 8-10 mmHg sabit diyastol sonu basıncı 100-110 mmHg arasında normal bir C57BL6 fare kalp LVDevP. Buna ek olarak, LabChart programı LV basınç dalgaları döngüsel ölçümlere göre kalp hızı ölçümü için izin verir. Yine, bu ölçü değişebilir ama kalpleri içsel fiyatla yenmek için izin verildiğinde tipik değerler 350-400 dak. Ancak, kalp, kalp hızı 420 bpm tutulur bir pacing sistemi kullanılarak standardize edilebilir. Buna ek olarak, kontraktilite (+ dP / dt) ve gevşeme (-dP/dt) önlemleri LV basınç dalgası ilk türevi kullanılarak tahmin edilebilir. Deneysel protokol sırasında, bir basınç-hacim ilişkileri dahil ederek Starling mekanizması değerlendirmek için kolaydır. Bu AG balon hacmi yavaş yavaş artar ve LVDevP yanı sıra EDP belirterek tarafından gerçekleştirilir. Bu değerler daha sonra Şekil 2&#39;de gösterilmiştir çizilebilir. Starling eğrisinin 8-10 mmHg bir EDP ulaşmak için gerekli hacim AG kamara boyut dolaylı bir fikir verebilir belirterek, en uygunudur. Bu kontroller ile karşılaştırıldığında daha büyük bir hacim genişlemiş kalpleri ihtiyaç duyarken, genellikle küçük bir balonun hacmi gerektirir hipertrofik kalpleri bantlama aort modelleri kullanılabilir. Tablo 1, perfüzyon protokolü sırasında edinilen temsilcisi kalp fonksiyonu verileri görüntüler. 31 P NMR spektrometresi phosphocreatine sinyalleri (PCR) ve ATP (γ-ATP, α-ATP, β-ATP) yanı sıra inorganik fosfat (Pi) Şekil 2&#39;de gösterildiği gibi üç fosfat sağlayacaktır. Bu zirveleri her Analizi eğri altında kalan alan için bir değer sağlar. ATP miktarı γ-ATP ve β-ATP bölgelerde ortalama tahmin edilmektedir. (Α-ATP NAD molekülleri toplam sinyal bilinmeyen bir kısmı katkıda çünkü kullanılmaz). Kalp enerjik durum PCr ve ATP alanlarda (ATP oranı PCr) katsayısı ile belirlenir. 1.7 birincil substrat olarak glikoz ile birlikte bir fare kalp - Bu değer genellikle 1,5 &#39;tir. 31 P NMR ATP veya PCr doğrudan tedbirler sağlamamakla birlikte, zirvelerin alanda örnek bileşikler içeren fosfor miktarı ile doğru orantılıdır. Bu sinyaller için değerler, diğer yöntemler kullanılarak tahmin edilebilir. Örneğin, kalplerin bir kohort yüksek performanslı sıvı kromatografi (HPLC) ile ATP doğrudan önlemler, ortalama bir konsantrasyon verim alınabilir. Bu değer daha sonra spektrumları gözlenen ortalama ATP alanlarda kalibre etmek için kullanılabilir. PCr konsantrasyonu ATP alana göre PCr alana göre hesaplanır. PCr sinyali göreceli inorganik fosfat kimyasal kayma (Pi) sinyali analiz ederek pH tahmin etmek de mümkündür. 1 farklı radyo sinyali dizileri, kreatin kinaz reaksiyon hızı veya ATP sentezi reaksiyon hızı da ölçülebilir. 2 <img src="/files/ftp_upload/2069/2069tab1.jpg" alt="Tablo 1" /> Tablo 1: İzole perfüze kalbinden Temel kalp fonksiyonu. LVDevP: sol ventrikül basıncı; LVEDP: sol ventrikül diyastol sonu basıncı; İK: kalp hızı; CHP oranı: basınç ürünü; + dP / dt: ilk türev LV basınç pozitif; -dP/dt: ilk türev LV basınç negatif, PP : perfüzyon basıncı; CF: koroner akım. <img src="/files/ftp_upload/2069/2069fig1.jpg" alt="Şekil 1" /> LabChart Pro yazılımı Şekil 1. Temsilcisi LV basınç dalgaları. <img src="/files/ftp_upload/2069/2069fig2.jpg" alt="Şekil 2" /> Şekil 2 kontrolü (düz çizgi) ve aort bantlı (noktalı çizgi) farelerde Temsilcisi Starling eğrileri. A) Sistolik fonksiyonu olarak artan LV hacimleri üzerinden LVDevP tarafından temsil LV balon birim tarafından belirlenir. B) LV balonun hacmi ile artan LV hacimleri üzerinden EDP tarafından temsil Diyastolik fonksiyonu olarak belirlenir. LVDevP: sol ventrikül basıncı (sistolik eksi diyastolik pressure); EDP: end-diastolik basınç. <img src="/files/ftp_upload/2069/2069fig3.jpg" alt="Şekil 3" /> Şekil 3. İzole perfüze fare kalp Temsilcisi 31 P NMR spektrumları. Nispeten küçük Pi zirve dikkat edin. Glikoz pirüvat veya yağ asitleri ile birlikte bir aerobik perfüze kalp, bu zirve düzeyde olmalıdır. Iskemi dönemlerde, bu pik artar PCr zirve azalır. Α-ATP zirve sağ omuz dikkat edin. Bu NAD moleküllerin katkıdır. Pi: inorganik fosfat; PCr: fosfokreatin; ATP: Adenozin trifosfat.

<ol>
	<li>Nascimben, L., Ingwall, J. S., Lorell, B. H., Pinz, I., Schultz, V., Tornheim, K., Tian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+for+increased+glycolysis+in+the+hypertrophied+rat+heart.">Mechanisms for increased glycolysis in the hypertrophied rat heart.</a> Hypertension. 44, 662-667 (2004).</li><li>Spindler, M., Saupe, K. W., Tian, R., Ahmed, S., Matlib, M. A., Ingwall, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+creatine+kinase+enzyme+kinetics+in+diabetic+cardiomyopathy.+A(31)P+NMR+magnetization+transfer+study+of+the+intact+beating+rat+heart.">Altered creatine kinase enzyme kinetics in diabetic cardiomyopathy. A(31)P NMR magnetization transfer study of the intact beating rat heart.</a> J Mol Cell Cardiol. 31, 2175-2189 (1999).</li><li>Ingwall, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorus+nuclear+magnetic+resonance+spectroscopy+of+cardiac+and+skeletal+muscles.">Phosphorus nuclear magnetic resonance spectroscopy of cardiac and skeletal muscles.</a> Am J Physiol. 242, H729-H744 (1982).</li><li>Ingwall, J. S., Javadpour, M. M., Miao, W., Hoit, B. D., Walsh, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=31P+NMR+spectroscopy+of+the+mouse+heart.">31P NMR spectroscopy of the mouse heart.</a> Cardiovascular physiology in the genetically engineered. , 151-163 (2002).</li><li>Luptak, I., Balschi, J. A., Xing, Y., Leone, T. C., Kelly, D. P., Tian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+contractile+and+metabolic+reserve+in+peroxisome+proliferator-activated+receptor-alpha-null+hearts+can+be+rescued+by+increasing+glucose+transport+and+utilization.">Decreased contractile and metabolic reserve in peroxisome proliferator-activated receptor-alpha-null hearts can be rescued by increasing glucose transport and utilization.</a> Circulation. 112, 2339-2346 (2005).</li><li>Yan, J., Young, M. E., Cui, L., Lopaschuk, G. D., Liao, R., Tian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+glucose+uptake+and+oxidation+in+mouse+hearts+prevent+high+fatty+acid+oxidation+but+cause+cardiac+dysfunction+in+diet-induced+obesity.">Increased glucose uptake and oxidation in mouse hearts prevent high fatty acid oxidation but cause cardiac dysfunction in diet-induced obesity.</a> Circulation. 119, 2818-2828 (2009).</li><li>Luptak, I., Shen, M., He, H., Hirshman, M. F., Musi, N., Goodyear, L. J., Yan, J., Wakimoto, H., Morita, H., Arad, M., Seidman, C. E., Seidman, J. G., Ingwall, J. S., Balschi, J. A., Tian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrant+activation+of+AMP-activated+protein+kinase+remodels+metabolic+network+in+favor+of+cardiac+glycogen+storage.">Aberrant activation of AMP-activated protein kinase remodels metabolic network in favor of cardiac glycogen storage.</a> J Clin Invest. 117, 1432-1439 (2007).</li><li>Xing, Y., Musi, N., Fujii, N., Zou, L., Luptak, I., Hirshman, M. F., Goodyear, L. J., Tian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+metabolism+and+energy+homeostasis+in+mouse+hearts+overexpressing+dominant+negative+alpha2+subunit+of+AMP-activated+protein+kinase.">Glucose metabolism and energy homeostasis in mouse hearts overexpressing dominant negative alpha2 subunit of AMP-activated protein kinase.</a> J Biol Chem. 278, 28372-28377 (2003).</li><li>Luptak, I., Yan, J., Cui, L., Jain, M., Liao, R., Tian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+effects+of+increased+glucose+entry+on+mouse+hearts+during+normal+aging+and+ischemic+stress.">Long-term effects of increased glucose entry on mouse hearts during normal aging and ischemic stress.</a> Circulation. 116, 901-909 (2007).</li><li>Tian, R., Abel, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+GLUT4-deficient+hearts+to+ischemia+underscore+the+importance+of+glycolysis.">Responses of GLUT4-deficient hearts to ischemia underscore the importance of glycolysis.</a> Circulation. 103, 2961-2966 (2001).</li><li>Liao, R., Jain, M., Cui, L., D'Agostino, J., Aiello, F., Luptak, I., Ngoy, S., Mortensen, R. M., Tian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac-specific+overexpression+of+GLUT1+prevents+the+development+of+heart+failure+attributable+to+pressure+overload+in+mice.">Cardiac-specific overexpression of GLUT1 prevents the development of heart failure attributable to pressure overload in mice.</a> Circulation. 106, 2125-2131 (2002).</li><li>Tian, R., Musi, N., D'Agostino, J., Hirshman, M. F., Goodyear, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+adenosine+monophosphate-activated+protein+kinase+activity+in+rat+hearts+with+pressure-overload+hypertrophy.">Increased adenosine monophosphate-activated protein kinase activity in rat hearts with pressure-overload hypertrophy.</a> Circulation. 104, 1664-1669 (2001).</li></ol>

Langendorff perfüze izole fare kalbinde 31 P NMR spektroskopisi güvenilir ve tekrarlanabilir bir veri sağlar. 3, 4 Ancak, LV balon aort ve ekleme kanülasyon Stabil kardiyak performans izin düzgün şekilde yapılması zorunludur NMR içinde iken tüp. Buna ek olarak, sıcaklık düzenlemesi uygun temel işlevleri yerine getirmek için her şeyden önemlidir. Sinyal gürültü oranı iyi analiz NMR spektrumları elde edilmesinde önemli bir faktör artmaktadır. Bu, optimal &quot;ayar&quot; sağlanması ve örnek &quot;layneri&quot; elde edilebilir. Protokol metninde belirtildiği gibi, kalbin takılması için önce standart bir örnek kullanımı kolaylaştırabilir. Yeterli büyüklükte bir &quot;örnek&quot; de yararlı olur. Az 100 mg ağırlığında Kalpler genellikle düşük PCr ve ATP sinyalleri bu nedenle alma süresi artar iyi fosfor spektrumları elde etmek için gerekli olacak sağlar. Kardiyak fonksiyon ve enerji ile ilgili ek bilgi toplamak için mevcut protokolü değiştirmek için çeşitli yollar vardır. Laboratuvarımızda, yağ asitlerinin farklı kombinasyonları (düşük ve yüksek konsantrasyonlarda), laktat, ketonlar, ve insülin varlığında içerebilir karışık substrat tamponlar ile kalpleri perfüze var. Perfüzyon tampon kararlı izotopların kullanılması (yani, 13 C yüzeylerde etiketli), TCA döngüsü etiketli asetil CoA göreceli katkısı substrat kullanımını tahmin etmek yeteneğine sahip bu uygulama için 5-7, biz gerçekleştirmek isotopomer 13 C3-ve 13 C4-glutamat 13 C NMR spektroskopisi ile analiz. Bu perfüzyon protokol sonunda kalp donma-sıkma ve dondurulmuş doku çıkarma performans gerektirir. Bu analiz, ayrı bir kurulum parametreleri ile farklı bir prob kullanımı gerektirdiği gibi ek bir deney olacak. Zamana bağımlı olarak, 31 P-NMR spektroskopisi kullanarak kalp 2-deoksiglukoz fosfat birikimi izleme sırasında diğer uygulamaları ikame tampon deoksiglukoz glikoz içerir . Bu yöntem, miyokard glukoz alımı ölçümü için sağlar. Buna ek olarak 7, 8, 6, kardiyak iskemi / reperfüzyon ve yüksek iş yükü zorluk oluşan perfüzyon protokolleri fonksiyonu ve enerji laboratuarımızda analiz etti. 8-10 Özet olarak, izole fare kalplerinde 31 P NMR spektroskopisi sofistike ekipman kullanımını gerektiren teknik olarak oldukça zor bir işlemdir . Ancak, verimi verileri kaldırılmıs fare modelleri fonksiyonu ve enerji analiz etmek isteyen araştırmacı için çok değerli. Laboratuvarımızda, bu tekniklerin, kalp fonksiyonu, enerji ve metabolizma streslerinin çeşitli sonuçları anlayışımız hayati edilmiştir 1, 11, 12

<table border="1">
<tbody>
<tr>
<td>Material Name</td>
<td>Type</td>
<td>Company</td>
<td>Catalogue Number</td>
</tr>
<tr>
<td>Magnesium Sulfate</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>M7506</td>
</tr>
<tr>
<td>EDTA</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>E1644</td>
</tr>
<tr>
<td>Potassium chloride</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>P4505</td>
</tr>
<tr>
<td>Sodium bicarbonate</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>S6297</td>
</tr>
<tr>
<td>Sodium chloride</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>S7653</td>
</tr>
<tr>
<td>Calcium chloride dihydrate</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>C5080</td>
</tr><tr>
<td>D-Glucose</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>G7528</td>
</tr><tr>
<td>Sodium Pyruvate</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>P2256</td>
</tr><tr>
<td>Bruker Ultrashield 600WB Plus</td>
<td>Equipment</td>
<td>Bruker</td>
<td>&nbsp;</td>
</tr><tr>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/ML866/" target="_blank">PowerLab 4/30</a></td>
<td>Equipment</td>
<td><a href="http://www.adinstruments.com" target="_blank">ADInstruments</a></td>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/ML866/" target="_blank">ML866/P</a></td>
</tr><tr>
<td><a href="http://www.adinstruments.com/products/software/research/LabChart-Software/" target="_blank">LabChart 6 Pro</a></td>
<td>Equipment</td>
<td><a href="http://www.adinstruments.com" target="_blank">ADInstruments</a></td>
<td><a href="http://www.adinstruments.com/products/software/research/LabChart-Software/" target="_blank">MLS260/6</a></td>
</tr><tr>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/FE224/" target="_blank">Quad Bridge Amp</a></td>
<td>Equipment</td>
<td><a href="http://www.adinstruments.com" target="_blank">ADInstruments</a></td>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/FE224/" target="_blank">ML224</a></td>
</tr><tr>
<td><a href="http://www.adinstruments.com/products/hardware/research/product/ML175/" target="_blank">STH Pump Controller</a></td>
<td>Equipment</td>
<td><a href="http://www.adinstruments.com" target="_blank">ADInstruments</a></td>
<td><a href="http://www.adinstruments.com/products/hardware/research/product/ML175/" target="_blank">ML175</a></td>
</tr><tr>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/ML172/" target="_blank">Minipuls 3 Peristaltic Pump</a></td>
<td>Equipment</td>
<td><a href="http://www.adinstruments.com" target="_blank">ADInstruments</a></td>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/ML172/" target="_blank">ML172</a></td>
</tr><tr>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/MLT0699/" target="_blank">Disposable BP Transducer</a></td>
<td>Equipment</td>
<td><a href="http://www.adinstruments.com" target="_blank">ADInstruments</a></td>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/MLT0699/" target="_blank">MLT0699</a></td>
</tr><tr>
<td>10mm NMR Sample Tube</td>
<td>Equipment</td>
<td>Wilmad LabGlass</td>
<td>513-7PP-7</td>
</tr><tr>
<td>Polyethylene tubing PE10</td>
<td>Equipment</td>
<td>Becton-Dickinson</td>
<td>427401</td>
</tr><tr>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/MLT844/" target="_blank">Physiological Pressure Transducer</a></td>
<td>Equipment</td>
<td><a href="http://www.adinstruments.com" target="_blank">ADInstruments</a></td>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/MLT844/" target="_blank">MLT844</a></td>
</tr><tr>
<td>Polyethylene tubing PE50</td>
<td>Equipment</td>
<td>Becton-Dickinson</td>
<td>427411</td>
</tr><tr>
<td>Micrometer syringe</td>
<td>Equipment</td>
<td>Gilmont Instruments</td>
<td>GS-1101</td>
</tr><tr>
<td>McPherson Forceps</td>
<td>Equipment</td>
<td>Miltex Inc.</td>
<td>18-949</td>
</tr><tr>
<td>Castraviejo microscissors</td>
<td>Equipment</td>
<td>Roboz Surgical Instruments</td>
<td>RS-5650</td>
</tr><tr>
<td>Neoptix Signal Conditioner</td>
<td>Equipment</td>
<td>Neoptix, Inc.</td>
<td>Reflex - 1</td>
</tr>	
</tbody>
</table>

assessment of cardiac function and energetics in isolated mouse hearts using 31p nmr spectroscopy

Kaldırılmıs fare modelleri, kardiyovasküler hastalık moleküler değişiklikler ve modeller arasında nedensel ilişkileri belirleyen güçlü araştırma araçları haline gelmiştir. Moleküler biyoloji sinyal yolağı önemli değişikliklerin tanımlanması gerekli olmasına rağmen, fonksiyonel önemi bir göstergesi değildir. Fizyolojisi fizyolojisi sağlam metabolitleri biyokimyasal değerlendirme ile birleştirerek, fonksiyon soru cevaplar sağlayabilir yenerek, kalp, kalp fonksiyonu ve enerji tam bir resim sağlar. Laboratuvarımızda yıl boyunca, bu görevi başarmak için nükleer manyetik rezonans (NMR) spektroskopisi ile birlikte izole kalp perfüzyonları kullanmıştır. Sol ventrikül fonksiyonu, kalp enerji yaparak ölçülür Langendorff modu izole kalp perfüzyonları tarafından değerlendirilir<sup&gt; 31</sup&gt; P perfüze kalplerinin manyetik rezonans spektroskopisi. Bu tekniklerin, phosphocreatine ve ATP düzeyleri ile birlikte kardiyak fonksiyon göstergeleri aynı anda kalpleri yenerek ölçülebilir yapabilirsiniz. Ayrıca, fizyolojik veya patolojik streslerinin tesis ise bu parametreler izlenebilir. Örneğin, iskemi / reperfüzyon veya yüksek iş yükü meydan protokoller kabul edilebilir. Bantlama aort veya kardiyak patolojisi diğer modelleri de apt. Varyantları protokol içinde ne olursa olsun, transgenik fare modelleri moleküler değişiklikler fonksiyonel ve enerjik önemi yeterince ilişkili enzim ve metabolik yollar yeni bakış açıları yol tarif edilebilir. Bu nedenle,<sup&gt; 31</sup&gt; P NMR spektroskopisi izole perfüze kalp kardiyovasküler hastalık hayvan modellerinde değerli bir araştırma tekniğidir.

Watch this Scientific Journal Video about Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy at JoVE.com

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

Langendorff-mode ile birlikte izole kalp perfüzyon<sup&gt; 31</sup&gt; P NMR spektroskopisi bir deney, biyokimya ve fizyoloji alanlarında birleştirir. Protokol dinamik ölçümü için yüksek enerjili fosfat içeriği ve fizyolojik fonksiyonu aynı anda izlerken kalp ciro sağlar. Doğru yapıldığında, bu kalp enerji değerlendirilmesinde değerli bir tekniktir.

Kalp Fonksiyonu ve Enerji Kullanımı İzole Fare Kalpler Değerlendirilmesi 31 P NMR Spektroskopisi

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

assessment-cardiac-function-energetics-isolated-mouse-hearts-using

Research

JoVE Journal

Medicine

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

26.0K Görüntüleme.  University of Washington School of Medicine. Langendorff-mode ile birlikte izole kalp perfüzyon 31 P NMR spektroskopisi bir deney, biyokimya ve fizyoloji alanlarında birleştirir. Protokol dinamik ölçümü için yüksek enerjili fosfat içeriği ve fizyolojik fonksiyonu aynı anda izlerken kalp ciro sağlar. Doğru yapıldığında, bu kalp enerji değerlendirilmesinde değerli bir tekniktir.

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

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited for studies of cardiac metabolism, which require the heart to perform work within the context of physiologic preload and afterload pressures. Neely introduced modifications to the Langendorff technique to establish appropriate left ventricular (LV) preload and afterload pressures3. The model is known as the isolated LV working heart model and has been used extensively to study LV performance and metabolism4-6. This model, however, does not provide a properly loaded right ventricle (RV). Demmy et al. first reported a biventricular model as a modification of the LV working heart model7, 8. They found that stroke volume, cardiac output, and pressure development improved in hearts converted from working LV mode to biventricular working mode8. A properly loaded RV also diminishes abnormal pressure gradients across the septum to improve septal function. Biventricular working hearts have been shown to maintain aortic output, pulmonary flow, mean aortic pressure, heart rate, and myocardial ATP levels for up to 3 hours8.
When studying the metabolic effects of myocardial injury, such as ischemia, it is often necessary to identify the location of the affected tissue. This can be done by imaging the fluorescence of NADH (the reduced form of nicotinamide adenine dinucleotide)9-11, a coenzyme found in large quantities in the mitochondria. NADH fluorescence (fNADH) displays a near linearly inverse relationship with local oxygen concentration12 and provides a measure of mitochondrial redox state13. fNADH imaging during hypoxic and ischemic conditions has been used as a dye-free method to identify hypoxic regions14, 15 and to monitor the progression of hypoxic conditions over time10.
The objective of the method is to monitor the mitochondrial redox state of biventricular working hearts during protocols that alter the rate of myocyte metabolism or induce hypoxia or create a combination of the two. Hearts from New Zealand white rabbits were connected to a biventricular working heart system (Hugo Sachs Elektronik) and perfused with modified Krebs-Henseleit solution16 at 37 &deg;C. Aortic, LV, pulmonary artery, and left &amp; right atrial pressures were recorded. Electrical activity was measured using a monophasic action potential electrode. To image fNADH, light from a mercury lamp was filtered (350&plusmn;25 nm) and used to illuminate the epicardium. Emitted light was filtered (460&plusmn;20 nm) and imaged using a CCD camera. Changes in the epicardial fNADH of biventricular working hearts during different pacing rates are presented. The combination of the heart model and fNADH imaging provides a new and valuable experimental tool for studying acute cardiac pathologies within the context of realistic physiological conditions.

The objective is to monitor the mitochondrial redox state of isolated hearts within the context of physiologic preload and afterload pressures. A biventricular working rabbit heart model is presented. High spatiotemporal resolution fluorescence imaging of NADH is used to monitor the mitochondrial redox state of epicardial tissue.

İzole Biventriküler ve NADH Floresans Görüntüleme Tavşan Kalpler Çalışma

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited ...

Tıp

Reduction in Left Ventricular Wall Stress and Improvement in Function in Failing Hearts using Algisyl-LVR

Injection of Algisyl-LVR, a treatment under clinical development, is intended to treat patients with dilated cardiomyopathy. This treatment was recently used for the first time in patients who had symptomatic heart failure. In all patients, cardiac function of the left ventricle (LV) improved significantly, as manifested by consistent reduction of the LV volume and wall stress. Here we describe this novel treatment procedure and the methods used to quantify its effects on LV wall stress and function.
Algisyl-LVR is a biopolymer gel consisting of Na+-Alginate and Ca2+-Alginate. The treatment procedure was carried out by mixing these two components and then combining them into one syringe for intramyocardial injections. This mixture was injected at 10 to 19 locations mid-way between the base and apex of the LV free wall in patients.
Magnetic resonance imaging (MRI), together with mathematical modeling, was used to quantify the effects of this treatment in patients before treatment and at various time points during recovery. The epicardial and endocardial surfaces were first digitized from the MR images to reconstruct the LV geometry at end-systole and at end-diastole. Left ventricular cavity volumes were then measured from these reconstructed surfaces.
Mathematical models of the LV were created from these MRI-reconstructed surfaces to calculate regional myofiber stress. Each LV model was constructed so that 1) it deforms according to a previously validated stress-strain relationship of the myocardium, and 2) the predicted LV cavity volume from these models matches the corresponding MRI-measured volume at end-diastole and end-systole. Diastolic filling was simulated by loading the LV endocardial surface with a prescribed end-diastolic pressure. Systolic contraction was simulated by concurrently loading the endocardial surface with a prescribed end-systolic pressure and adding active contraction in the myofiber direction. Regional myofiber stress at end-diastole and end-systole was computed from the deformed LV based on the stress-strain relationship.

This article describes procedures for implanting a novel hydrogel in failing hearts and quantifying its effect on left ventricular wall stress and function. These procedures have been successfully applied in dogs and humans.

Algisyl-LVR kullanarak Hearts başarısız Fonksiyon Sol Ventrikül Duvar Stres ve İyileştirme azalma

Injection of  Algisyl-LVR, a treatment under clinical development, is intended to treat  patients with dilated cardiomyopathy. This treatment was recently ...

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery (SALLI) MRI in Rats

Small animal magnetic resonance imaging is an important tool to study cardiac function and changes in myocardial tissue. The high heart rates of small animals (200 to 600 beats/min) have previously limited the role of CMR imaging. Small animal Look-Locker inversion recovery (SALLI) is a T1 mapping sequence for small animals to overcome this problem 1. T1 maps provide quantitative information about tissue alterations and contrast agent kinetics. It is also possible to detect diffuse myocardial processes such as interstitial fibrosis or edema 1-6. Furthermore, from a single set of image data, it is possible to examine heart function and myocardial scarring by generating cine and inversion recovery-prepared late gadolinium enhancement-type MR images 1.
	The presented video shows step-by-step the procedures to perform small animal CMR imaging. Here it is presented with a healthy Sprague-Dawley rat, however naturally it can be extended to different cardiac small animal models.

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery SALLI MRI in Rats

Contrast enhanced cardiac magnetic resonance (CMR) imaging allows comprehensive in vivo assessment of the heart in small animal models of cardiovascular disease. Here we detail the procedures to perform CMR imaging, reconstruction and analysis using Small Animal Lock-Locker Inversion Recovery (SALLI) in rats.

Küçük Hayvan Bak-soyunma Ters Kurtarma (SALLI) Sıçanlarda MRI kullanarak Kalp Fonksiyonu ve Miyokard Morfoloji Değerlendirilmesi

Small animal magnetic resonance  imaging is an important tool to study cardiac function and changes in myocardial  tissue. The high heart rates of small ...

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

Transtorasik Ekokardiyografi ile Pulmoner Arter Konstrüksiyon Fare Modeli sağ ventrikül yapı ve fonksiyonlarının değerlendirilmesi

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

Langendorff-perfüze sıçan Hearts indüksiyon ve İskemi-reperfüzyon Hasarı Değerlendirilmesi

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

İnsan yağ dokusu mikrovasküler işlevini kullanarak Videomicroscopy değerlendirilmesi

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

Fare modelleri Glomerular hastalığı böbrek fonksiyonlarının değerlendirilmesi

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation time and increases the risk of adverse post-transplantation outcomes. Moreover, the static nature of CS does not allow for organ evaluation or intervention during the preservation interval. Normothermic ex situ heart perfusion (ESHP) is a novel method for preservation of the donated heart that minimizes cold ischemia by providing oxygenated, nutrient-rich perfusate to the heart. ESHP has been shown to be non-inferior to CS in the preservation of standard-criteria donor hearts and has also facilitated the clinical transplantation of the hearts donated after the circulatory determination of death. Currently, the only available clinical ESHP device perfuses the heart in an unloaded, non-working state, limiting assessments of myocardial performance. Conversely, ESHP in working mode provides the opportunity for comprehensive evaluation of cardiac performance by assessment of functional and metabolic parameters under physiologic conditions. Moreover, earlier experimental studies have suggested that ESHP in working mode may result in improved functional preservation. Here, we describe the protocol for ex situ perfusion of the heart in a large mammal (porcine) model, which is reproducible for different animal models and heart sizes. The software program in this ESHP apparatus allows for real-time and automated control of the pump speed to maintain desired aortic and left atrial pressure and evaluates a variety of functional and electrophysiological parameters with minimal need for supervision/manipulation.

Normothermic ex situ heart perfusion (ESHP), preserves the heart in a beating, semi-physiologic state. When performed in a working mode, ESHP provides the opportunity to perform sophisticated assessments of donor heart function and organ viability. Here, we describe our method for myocardial performance evaluation during ESHP.

Normothermic Ex Situ kalp perfüzyon çalışma modu: değerlendirme kardiyak fonksiyon ve metabolizma

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation ...

Echocardiographic Assessment of Cardiac Anatomy and Function in Adult Rats

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional imaging to study the cardiac anatomy of the left ventricle and M-mode echo to assess its dimensions. However, this could limit a comprehensive study. Herein, we describe a protocol that allows an assessment of the heart chamber size, left ventricular function (systolic and diastolic) and valvular function. A conventional medical ultrasound machine was used in this protocol and different echo views were obtained through left parasternal, apical and suprasternal windows. In the left parasternal window, the long and short axis were acquired to analyze left chamber dimensions, right ventricle and pulmonary artery dimensions, and mitral, pulmonary and aortic valve function. The apical window allows the measurement of heart chamber dimensions and evaluation of systolic and diastolic parameters. It also allows Doppler assessment with detection and quantification of heart valve disturbances (regurgitation or stenosis). Different segments and walls of the left ventricle are visualized throughout all views. Finally, the ascending aorta, aortic arch, and descending aorta can be imaged through the suprasternal window. A combination of ultrasound imaging, Doppler flow and tissue Doppler assessment have been obtained to study cardiac morphology and function. This represents an important contribution to improve the assessment of cardiac function in adult rats with impact for research using these animal models.

A non-invasive protocol for transthoracic echocardiography assessment of cardiac anatomy and function for adult rats is presented in the current study. The heart valves, all four cardiac chambers and the ascending aorta, aortic arch and descending aorta are studied in detail.

Erişkin Sıçanlarda Kardiyak Anatomi ve Fonksiyonun Ekokardiyografik Değerlendirilmesi

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional ...

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 ...