Bu araştırma Eğitim, Bilim ve Teknoloji Bakanlığı (2013068067) tarafından finanse Kore Ulusal Araştırma Vakfı (ÇKS) ile Temel Bilimler Araştırma Programı tarafından desteklenen; Eğitim, Bilim ve Teknoloji, Seul Ulusal Üniversitesi Hastanesi, Hipertansiyon Kore Derneği&#39;nin (2013), SK Telecom Araştırma Fonu Kore Bakanlığı Beyin Kore 21 Yüksek Lisans Programı tarafından (no. 3420130290) ve Çin Ulusal Doğa Bilimleri Vakfı (NSFC 31460265; NSFC 81260035).

protokol Sağlık BM Ulusal Sağlık Enstitüleri tarafından yayınlanan Laboratuvar Hayvanları Bakım ve Kullanım Kılavuzu uygundur (NIH Yayın No. 85-23, 1996 revize). Bu Seul Ulusal Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Komitesi (IACUC) (IACUC onayı No .: SNU-101213-1) tarafından onaylanmıştır. 1. Tampon Hazırlama (Tablo Malzemeleri ve Ekipmanları) <ol><li> MM (deney gününde taze yalıtım çözeltisi 300 ml hazırlayın: NaCl, 135; KCI, 5.4; MgCl2, 3.5; glukoz, 5; HEPES, 5; Na 2 HPO 4, 0.4, taurin, 20; pH 7.4 NaOH ile birlikte). </li><li> MM (deney gününde taze bir depolama solüsyonu 100 ml hazırlayın: NaCI 120; KCI 5,4; MgSO 4 5&#39;tir; CaCl2 0.2; sodyum piruvat 5&#39;tir; 5.5 glikoz, taurin 20, HEPES 10, manitol 29, pH 7.4 ) NaOH ile. </li><li> MM perfüzyon çözeltisi 1L (hazırlayın: NaCl, 141.4; KCI, 4; NaH 2 PO 4, 0.33; MGCL2, 1 &#39;dir; HEPES, 10; glukoz, 5.5; CaCl2, 1.8; manitol, 14.5; NaOH ile pH 7.4). </li><li> Izolasyon 25 ml kollajenaz (1 mg / ml), proteaz (0.1 mg / ml), sığır serum albümini (BSA, 1.67mg / ml) ve Ca 2 + (0.05 mM) ilave edilerek kolajenaz çözeltisi 30 ml 1 Hazırlama çözüm. </li><li> Yalıtım çözeltisi 16.7 ml (1 mg / ml), BSA (1.67 mg / ml) ve Ca + 2 (0.05 mM) kolajenaz ekleyerek kolajenaz çözeltisi 2 20 ml hazırlayın. </li><li> yalıtım çözeltisi 40 ml BSA 0.4 g eklenerek BSA çözeltisi 40 ml hazırlayın. 10 ml ve iki bardak içine 30 ml ayrı. : Ca BSA çözeltisi 2 + 30 ml ilave edilir, böylece BSA çözeltisi nihai Ca2 + konsantrasyon 1 mM&#39;dir. </li></ol> Sol Ventriküler (LV) miyositler İzolasyonunda 2. Hazırlık <ol><li> hazırlanması ve izolasyon odasına hayvan tesis temiz ulaşım kafeslerde 8-12 haftalık, erkek Sprague-Dawley (SD) sıçanları aktarın. </li><li> H37 o C iki su banyoları yemek Not: Su için bir su banyosu kullanın - ceketli rezervuar ve Langendorff perfüzyon sistemi perfüzyon tüpleri. Tek miyositleri ayırmak amacıyla miyokard doku gövdelerine kışkırtmak diğer su banyosu kullanın. </li><li> Sırasıyla, 30 ml ve 20 ml hacim yapmak için 5 ml ve kolajenaz çözeltisi 1 ve 16.7 ml kolagenaz çözeltisi 2 25 ml (yaklaşık olmayan 2+) BSA çözeltisi 3.3 ml. </li><li> Langendorff perfüzyon sistemine sırasıyla (Şekil 1A) izolasyon çözeltisi (100 mi) ve sütun 1 (Col 1) ile sütun 2 kolajenaz çözeltisi 1 (30 mi) (Col 2) ekleyin. </li><li> Langendorff perfüzyon sistemine (Şekil 1A) oksijen birleştirme borusu yoluyla, Sütun 1 ve Col.2 yalıtım solüsyonu ve kolajenaz çözeltisi 1 oksijen. Benzer şekilde, oxygenate kollajenaz çözüm 2 ve üzeri% 100 O 2 ile sallayarak su banyosunda BSA çözeltisiOksijen bağlantı borusu. </li></ol> LV miyosit 3. İzolasyon <ol><li> Pentobarbital sodyum (30 mg / kg) intraperitonal enjeksiyonu ile SD sıçan anestezisi ve kıstırma ayak ve çekme yansıma eksikliği anestezi durumunu teyit. </li><li> Bir diseksiyon tepsisine sıçan taşıyın. yatar pozisyonda, bant ile vücudun kenarlarına dört ayağı sabitleyin. </li><li> kalbi zarar vermemeye sağlanması, göğüs açmak için cerrahi makas ile göğüs orta eksenel kesiler uygulayın. Bağlantı gemiler (örneğin, üst ve alt vena kava, pulmoner damarları ve aort) ve perikardiyal zarından 13 den kalbi incelemek için temiz makas başka bir çift kullanın. </li><li> Yeterli uzunlukta aort bir kısmını bırakın (5-8 mm), ince forseps ile aort kelepçe ve hızla 1 dakika (Şekil 1A) içinde Langendorff perfüzyon sistemi kanül monte edin. aorta üzerinde sıkıca dikiş ipliği 4/0 kravat. </li><li&gt; Sütun 1 vananın açma ve 10 dakika boyunca önceden ısıtılmış ve oksijenli izolasyon solüsyonu (perfüzyon oranı: 12-14 ml / dak) ile izole kalbi serpmek. , Col 1 vanayı kapatınız Sütun 2 valfi açmak ve 8 kollajenaz çözüm 1 ile kalbi serpmek - 10 dk. </li><li> Aorta keserek sindirilmiş kalbi ayırın ve taze izolasyon solüsyonu (Şekil 1Bi) içeren balona forseps ile aort tutarak aktarın. Daha küçük parçalara (~ 22 mm, Şekil 1Bii) içine (septum dahil) LV serbest duvar en kesmek için ince makas ve forseps kullanın. </li><li> Önceden ısıtılmış ve oksijenli taze kolajenaz çözeltisi 2 (Şekil 1Biii) ihtiva eden bir şişe içine parçaları aktarın. 10 dakika süre ile çalkalanır. miyosit içeren kollajenaz çözüm 2 oksijen teslim tutun. </li><li> (BSA çözeltisi içeren - 2+ bir emme ampul ile damlatıcıları kullanarak 10 ml santrifüj tüpüne süspansiyon ve Ca ekleyin - miyosit TaşıHacim olarak 1: 1). 2 dakika süreyle 30 g Santrifüj ve süpernatant atın. BSA çözeltisi 5 ml miyosit pelet yeniden askıya. Santrifüj süpernatantı atmak ve. </li><li> Miyosit pelet dağılması ve oda sıcaklığında (Şekil 1Biv-VI) önceden oksijenli bir depolama solüsyonu 10 ml miyositleri tutun. </li><li> şişede kalan LV dokusu ile - prosedürleri (3.9 3.7 adımlar) tekrarlayın. LV dokusunun çoğu miyositler iyi bir verim elde etmek için kaybolana kadar tekrar - (3.9 3.7 adımlar) prosedürleri tekrarlayarak tutun: Not. </li><li> Deneylerin sonuna kadar oda sıcaklığında 6-8 saat süreyle depolama çözümü myositler tutun. </li></ol> Hücre içi Ca 2 Geçici ve miyosit daralma 4. Eşzamanlı Ölçümleri <ol><li> asetoksimetilester Fura-2 AM (2 mcM) ile yük LV miyositler. Not: Karanlık bir tüp (Şekil 1Bvii) yüklenen miyositler tüm yükleme prosedürleri ve deney gerçekleştirin. <ol><li> centr10 saniye boyunca 2000 x g&#39;de miyosit süspansiyonu (1 mi) ifuge. Süpernatantı atın ve düşük Ca 2 + konsantrasyonu (250 uM, Masa Malzemeleri ve Ekipmanları) ile Tyrode çözeltisi 1 ml miyosit pelet yeniden askıya. </li><li> Yavaşça miyosit süspansiyon, dispers, Fura-2AM ve poloksamer 407 (2 ul) ekleyin ve oda sıcaklığında karışımın tutulması - 15 dakika boyunca (20 ila 24 ° C) (Şekil 1Bvii). </li><li> Santrifüj Karışım 5 saniye için, yüzer tabaka atılır ve 500 uM Ca2 + içeren perfüzyon çözeltisi 1 ml miyosit pelet dağılır. 10 dakika sonra, 5 saniye için karışım santrifüj ve supernatant atın. </li><li> Taze perfüzyon çözeltisi (500 uM Ca2 +, 1 mi) ilave edilir ve, Fura-2AM tutulması - kayıtları için bu çözeltiye yüklenen miyosit pelet. </li></ol></li><li> LV miyosit kasılması ve hücre içi Ca ölçümü 2 + <ol><li> kaydetmeden önce, bir geçiyor perfüzyon tüpü doldurmakbir Tyrode çözeltisi ile su gömleği 36 o C önceden ısıtılmış </li><li> 8 dakika - 5 için ters bir floresan mikroskop odasının yüklenen LV miyosit süspansiyon - Bir Fura 2 AM birkaç damla koyun. Yavaş yavaş Tyrode çözeltisi (2.2 mL / dakika) serpmek. </li><li> alan stimülasyonu (2 Hz) başlatmak için dijital stimülatörü ön panelinde bulunan &quot;start&quot; düğmesine basın. Not: çıkış gerilimini (10 V, 5 msn süre) bölmenin her iki yanında yer alan platin teller üzerinden bölme miyosit uygulanır. <ol><li> stabil olduğu sözleşme Seç miyositler kayıt (hiper veya hipo-daralma göstererek olmayanlar). </li></ol></li><li> video tabanlı sarkomer algılama sisteminin yatay pozisyonda seçim miyosit ayarlayın ve sarkomer optimum görüntü elde etmek için mikroskop odağı ayarlayın. net sarkomer açıkça ortalamanın kadar gözlenir hangi alanda mor dikdörtgen kutu yerleştirinsarkomer uzunlukları (kırmızı tepe) bir tekil sivri bir tepe (Şekil 2A, alt resim) gösterir. </li><li> Alan stimülasyonu (Şekil 2A ve B) cevaben sarkomer uzunluğunun değişiklikleri kaydedin. Not: 1 içinde yüklü miyositleri kullanın - 2 saat. </li><li> Video tabanlı sarkomer algılama sistemine alan miyosit (Şekil 2A) boyutu böylece kameranın diyafram ayarlayın. 510 nm (satın alma frekansı 1000 Hz) 360 nm / 380 nm eksitasyon ve emisyon ile miyosit uyarır. Tutanak sarkomer kısalma ve alan uyarımı (Şekil 2B) ile Fura2 AM oranı. </li></ol></li></ol> Myofilament Ca 2+ Hassasiyet 5. Değerlendirme <ol><li> Ölçülen değer ile hem (- (20 izleri 10) ve aynı miyosit ve sarkomer uzunluğu vs Fura-2 oranı faz-düzlem döngü arsa sarkomer uzunlukları ve kararlı durumda 2 + transientler Ca ortalamas ve delta değişiklikleri; Şekil 2C). </li><li> Her arsa,% 50 rahatlama de Fura -2 oranı (EC 50, Şekil 2C) tanımlar. Döngü ve her müdahale EC 50 hem karşılaştırın. </li></ol>

<ol>
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The authors declare that they have no competing financial interests.

Burada, tek izole kardiyak miyosit içinde Myofilament Ca 2+ duyarlılık değişiklikleri değerlendirmek için protokoller tanımlar ve bu elektrofizyolojik özellikleri yanında parametreyi, hücre içi Ca 2 + transientler ve Myofilament dinamiklerini ölçmek önemini vurgulamaktadır. bir ya da parametrelerin iki kayıtları kalp kasılma ve gevşeme altında yatan mekanizmaları izah olmayabilir olmasıdır. Miyosit daralma ve hücre içi Ca2 + profil ayrı ayrı 1 ölç...

Kardiyak eksitasyon-kasılma (EC) bağlantı miyokard, yani kalbin 1,2 kasılma fonksiyonu mekanik özelliklerini analiz etmek için temel düzenidir. AK bağlantı sarkolemmal iyon kanallarının faaliyetleri ikincil membran depolarizasyon tarafından başlatılan (örneğin, yama-kelepçe teknikleri ile ölçülebilir voltaj kapılı Na + kanal). (Nanomolar gelen nM sitozolik Ca 2 + konsantrasyonunun artması ryanodin reseptörleri (RyRs) aracılığıyla voltaj bağımlı L-tipi sonraki aktivasyonu Ca 2 + kanal LTCCs aracılığıyla 2+ akını Ca toplu tetikler (LTCCs) ve Ca 2+ açıklaması, ) (uM) seviyesini mikromolar. Sitosolik Ca2 + böyle bir artış, ince filamanların troponin C (TNC) bağlanabilen Ca2 + teşvik eder ve ortaya çıkarır filament kompleks yapı değişiklikleri, aktin-miyosin etkileşimin kolaylaştırmak ve myocardi ulaşıral 3 daralma. Tersine, sitozolik Ca 2 + SR Ca 2 + -ATPase&#39;ın ile sarkoplazmik retikulum (SR) (SERCA2a) geri re-Alınan veya Na + / Ca 2+ eşanjör ve plazmalemmal yoluyla miyosit dışarı ekstrüde edilir Ca + 2 ATPaz 1,2. Sonuç olarak, sitozolik Ca düşüş 2+ aktin-miyozin ve miyosit rahatlama 1-3 ayrışmasından kaynaklanan özgün durumuna ince filamentler yapısal değişiklikleri başlatır. Çoğu memeli kalp hücrelerinin 1 sitosolik Ca2 + kaldırılması% 90 - Bu şemada, SERCA2a aktivitesi, genel olarak 70 oluşturmaktadır miyokardiyal gevşeme hızını belirlemek için kabul edilir. Vb LTCC, RYR ve SERCA2a, böyle anormal Ca 2 + işleme gibi hastalıklı kalp 1-4 bozulmuş kontraktilite ve dinlenmek için birincil mekanizma olarak kabul edilmiştir. 2+ toplam hücre içi Ca yaklaşık% 1 EC bağlantı hesaplarında haberci ve Ca 2+ çoğunluğu olarak işlev hücre içi Ca 2 + tamponlar 5,6 bağlıdır serbest sitozolik Ca 2 +. Bu durum, çeşitli Ca 2 + tamponlar, SR, örneğin, membran fosfolipidleri, ATP, fosfokreatin, kalmodulin, parvalbumin, myofibril TNC miyosin, SERCA2a ve calsequestrin kardiyak miyositlerde bol olduğu gerçeğidir. 5,6,7. Aralarında, SERCA2a ve TNC baskın Ca2 + tamponlar 5,6,7 vardır. Ayrıca, Ca 2 + onun tamponlar bağlanmasını seğirme sırasında dinamik bir süreçtir ve ek nedeni bağlayıcı Ca değişikliği 2+ (örneğin, 2+ Ca% 30-50 2+ transientler 7 TNC bağlanır ve Ca sırasında ondan ayırmak) kons 2+ hücre içi Ca değişiklikler de, sitoplazmada serbest Ca 2 + sonuçlarını &quot;serbest&quot;entration. Sonuç olarak, hücre içi Ca 2 + düzeyinin pertürbasyon kasılma disfonksiyonu ve aritmi 8,9 öncüleri olan anormal Myofilament hareketleri, neden olur. (Fizyolojik ve patolojik hem de) pek çok faktör Myofilament Ca 2 + tamponlama ve Myofilament Ca 2+ duyarlılığını 8-10 etkileyen Myofilament proteinlerin post-transkripsiyonel modifikasyonları, kaynakları olabilir. Son zamanlarda, bu Ca 2 + transientler, anormal Ca 2 + bırakma ve aritmiler 8 duraklamaya bağlı güçlendirilmesini tetikleme, Myofilament proteinleri mutasyonlar Ca 2 + bağlayıcı afinite ve hücre içi Ca2 + taşıma artış olduğu bildirilmiştir. Bu anlayış doğrultusunda, biz de yüksek sistolik ve diyastolik Ca 2 + düzeyleri ile ilişkili nöronal nitrik oksit sentaz up-regülasyonu sekonder hipertansif sıçan kalplerinde o Myofilament Ca 2 + duyarsızlaştırma göstermiştirSırayla, Ca 2 + bağımlı inaktivasyon 12 LTCC açığı artırmaktadır 11. Bu nedenle, Myofilament Ca 2+ duyarlılığı hücre içi Ca2 + homeostasis ve miyosit kasılma fonksiyonu bir &quot;aktif&quot; düzenleyicisidir. Bu Myofilament ve Ca arasındaki etkileşimleri analiz etmek için gerekli hale gelmiştir 2+ miyosit AK kavraması ve kalp fonksiyonlarının ayrıntılı bir araştırma için proteinleri ele. Burada, izole kardiyak miyositlerdeki Myofilament Ca 2+ duyarlılık değişiklikleri değerlendiren bir protokol açıklar. Hücre içi Ca 2 + profilinin kapsamlı bir analiz, Myofilament Ca 2+ duyarlılık ve daralma yeni mekanizmaları altında yatan miyokard mekaniği ortaya çıkarmak olacaktır.

<table><tbody><tr><td>Sprague Dawley rat</td><td>Koatech</td><td></td><td>8-12 weeks</td></tr><tr><td>Pentobarbital Sodium</td><td>Hanlim Pharmaceutical (Korea)</td><td>AHN901</td><td>Insurance code:645301220</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9625</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P4504</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>S8282</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>Glucose</td><td>Sigma</td><td>G8270</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Biosesang</td><td>C2002</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Biosesang</td><td>M2001</td><td></td></tr><tr><td>Mannitol</td><td>Sigma</td><td>M4125</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>M5921</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Sigma</td><td>P2256</td><td></td></tr><tr><td>Taurine</td><td>Merck</td><td>8.08616.1000</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&amp;nbsp;&lt;/sub&gt;</td><td>Sigma</td><td>71649</td><td></td></tr><tr><td>Bovine Fetal Albumin</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>Collagenase Type 2</td><td>Worthington</td><td>LS004177</td><td></td></tr><tr><td>Protease</td><td>Sigma</td><td>P6911</td><td></td></tr><tr><td>Fura-2 (AM)</td><td>Molecular Probes</td><td>F1221</td><td></td></tr><tr><td>Pluronic F127 20% solution in DMSO</td><td>Invitrogen</td><td>P3000MP</td><td></td></tr><tr><td>Shaking Water Bath</td><td>Chang Shin Scientific</td><td>Model: C-108</td><td></td></tr><tr><td>IonWizard Softwae Suite</td><td>IonOptix Ltd</td><td>Experimental Builder</td><td>Acquisition and Analysis of EC Coupling Data in Myocytes</td></tr><tr><td>Myocyte Calcium &amp;amp; Contractility Recording System</td><td>IonOptix Ltd</td><td></td><td></td></tr><tr><td>Circulating Water Bath</td><td>BS-Tech</td><td>BW2-8</td><td></td></tr><tr><td>Myocyte Fluorescence Microscope</td><td>Nikon</td><td>DIATPHOTO 200</td><td></td></tr><tr><td>MyoCam-S Power</td><td>IonOptix</td><td></td><td></td></tr><tr><td>Fluorescence &amp;amp; Video Detection</td><td>IonOptix</td><td>MyoCam-S</td><td></td></tr><tr><td>CFA300</td><td></td><td></td><td></td></tr><tr><td>PMT400</td><td></td><td></td><td></td></tr><tr><td>Fluorescence &amp;amp; System Interface</td><td>IonOptix</td><td>FSI700</td><td></td></tr><tr><td>Excitation Light Source</td><td>IonOptix</td><td>mSTEP</td><td></td></tr><tr><td>High intensity ARC Lamp Power supply</td><td>Cairn Reseach</td><td></td><td></td></tr><tr><td>Filter wheel controller</td><td>IonOptix</td><td>GB/MUS200</td><td></td></tr><tr><td>Digital Stimulator</td><td>Medical Systems Corportion</td><td>S-98 Mutimode</td><td></td></tr><tr><td>&lt;strong&gt;Compositions of Experimental Solutions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>Isolation Solution (pH: 7.4, NaOH)</td><td></td><td></td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9625</td><td>Concentration (mmol) 135</td></tr><tr><td>KCl</td><td>Sigma</td><td>P4504</td><td>Concentration (mmol) 5.4</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td>Concentration (mmol) 5</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G8270</td><td>Concentration (mmol) 5</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Biosesang</td><td>M2001</td><td>Concentration (mmol) 3.5</td></tr><tr><td>Taurine</td><td>Sigma</td><td>CB2742654</td><td>Concentration (mmol) 20</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&amp;nbsp;&lt;/sub&gt;</td><td>Sigma</td><td>71649</td><td>Concentration (mmol) 0.4</td></tr><tr><td>Storage Solution (pH: 7.4, NaOH)</td><td></td><td></td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9625</td><td>Concentration (mmol) 120</td></tr><tr><td>KCl</td><td>Sigma</td><td>P4504</td><td>Concentration (mmol) 5.4</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td>Concentration (mmol) 10</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G8270</td><td>Concentration (mmol) 5.5</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Biosesang</td><td>C2002</td><td>Concentration (mmol) 0.2</td></tr><tr><td>Mannitol</td><td>Sigma</td><td>M4125</td><td>Concentration (mmol) 29</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>M5921</td><td>Concentration (mmol) 5</td></tr><tr><td>Sodium Pyruvate</td><td>Sigma</td><td>P2256</td><td>Concentration (mmol) 5</td></tr><tr><td>Taurine</td><td>Sigma</td><td>CB2742654</td><td>Concentration (mmol) 20</td></tr><tr><td>Perfusion Solution (Tyrode solution, pH: 7.4, NaOH)</td><td></td><td></td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9625</td><td>Concentration (mmol) 141.4</td></tr><tr><td>KCl</td><td>Sigma</td><td>P4504</td><td>Concentration (mmol) 4</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>S8282</td><td>Concentration (mmol) 0.33</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td>Concentration (mmol) 10</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G8270</td><td>Concentration (mmol) 5.5</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Biosesang</td><td>C2002</td><td>Concentration (mmol) 1.8&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; For Fura 2AM loading, CaCl2 concentrations are 0.25 mM and 0.5 mM</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Biosesang</td><td>M2001</td><td>Concentration (mmol) 1</td></tr><tr><td>Mannitol</td><td>Sigma</td><td>M4125</td><td>Concentration (mmol) 14.5</td></tr><tr><td>Collangenase Solution 1</td><td></td><td></td><td></td></tr><tr><td>Isolation Solution (30mL)</td><td></td><td></td><td></td></tr><tr><td>Bovine Fetal Albumin (BSA solution 5 ml)</td><td></td><td></td><td>Concentration (mmol) 1.67 mg/mL</td></tr><tr><td>Collagenase Type 2</td><td>Worthington</td><td>LS004177</td><td>Concentration (mmol) 1 mg/mL</td></tr><tr><td>Protease</td><td>Sigma</td><td>P6911</td><td>Concentration (mmol) 0.1 mg/mL</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Biosesang</td><td>C2002</td><td>Concentration (mmol) 0.05 mM</td></tr><tr><td>Collangenase Solution 2</td><td></td><td></td><td></td></tr><tr><td>Isolation Solution (20mL)</td><td></td><td></td><td></td></tr><tr><td>Bovine Fetal Albumin (BSA solution 3.3 mL)</td><td></td><td></td><td>Concentration (mmol) 1.67 mg/mL</td></tr><tr><td>Collagenase Type 2</td><td>Worthington</td><td>LS004177</td><td>Concentration (mmol) 1 mg/mL</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Biosesang</td><td>C2002</td><td>Concentration (mmol) 0.05 mM</td></tr><tr><td>BSA solution</td><td></td><td></td><td></td></tr><tr><td>Isolation Solution (40mL)</td><td></td><td></td><td></td></tr><tr><td>Bovine Fetal Albumin</td><td>Sigma</td><td>A7906</td><td>Concentration (mmol) 400 mg</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Biosesang</td><td>C2002</td><td>Concentration (mmol) 1mM</td></tr></tbody></table>

assessment of myofilament ca2+ sensitivity underlying cardiac excitation-contraction coupling

Kalp yetmezliği ve kardiyak aritmi, dünya çapında mortalite ve morbidite nedenlerinin başında gelmektedir. Ancak, hastalıklı kalbinde patogenezi ve miyokard arıza mekanizması tam açıklığa kavuşturulması kalır. Son zorlayıcı kanıtlar Myofilament Ca 2+ duyarlılık değişiklikler kardiyak miyositler hücre içi Ca 2 + homeostazı ve iyon kanal faaliyetlerini etkileyen gösteriyor sağlıklı ve hastalıklı kalpleri kardiyak aksiyon potansiyeli ve daralma sorumlu temel mekanizmalar. Gerçekten de, (örneğin, Na +, Ca 2+ ve K + kanalları ve Na + -Ca 2+ değiştirici) ve hücre içi Ca 2 + taşıma proteinleri (örneğin., Ryanodin reseptörleri ve Ca kardiyak aksiyon potansiyelleri altında yatan iyon kanallarının faaliyetleri ve taşıyıcılar sarkoplazmik retikulum (SERCA2a) ya da fosfolamban&#39;ın ve fosforilasyon) 2 + ATPaz geleneksel katı değerlendirme için ölçülürKalp eksitasyon-kasılma (EC) bağlantının temel mekanizmaları yedik. Myofilament kasılma ve gevşeme fonksiyonel birimi ve Myofilament Ca 2 + duyarlılığı myofibril performans uygulanmasında zorunlu ise zarı ve hücre içi Ca + 2 değişiklikler iki elektriksel aktivitesindeki AT kuplaj başlatma sinyalleridir. Bu çalışmanın odak noktası olmadıkça Bununla birlikte, az sayıda çalışma miyokardın fonksiyonel analiz içine Myofilament Ca 2+ duyarlılığını içermektedir. Burada, sarkomer kısalma / yeniden uzaması ve Fura-2 AM (oranlı metrik algılama) ve sıçan kalpleri kardiyak miyozitlerde Myofilament Ca 2+ duyarlılık değişiklikleri değerlendirmek kullanarak hücre içi Ca 2 + seviyesini ölçen bir protokol açıklar. Temel amacı Ca 2+ duyarlılık mekanik analiz için AK bağlantı dikkate alınması gerektiğini Myofilament vurgulamaktır. i kapsamlı bir soruşturmaöteleme ve terapötik değeri daha etkili stratejiler tasarlamak için değerli bilgiler sağlayacaktır sağlıklı ve hastalıklı kalplerde miyosit kontraktilite altında yatan kanalları, iyon taşıyıcılar, hücre içi Ca 2 + taşıma ve Myofilament Ca 2+ hassasiyeti üzerinde.

LV miyositler normal ve hipertansif sıçan kalpleri izole edilmiştir. Alan uyarılmasına tepki olarak açık bir şeritlerin (sarkomer temsil eder) ve sabit kasılmalarla çubuk şekilli miyositler uygun miyositler olarak kabul edilir ve kayıt (Şekil 2A) için seçilir. F ŞEKIL 2A&#39;da gösterilen örnekte, Fura 02:00 AG miyosit yatay konumlandırılmış ve miyosit kayıt alanı ve en az arka bölgenin en dahildir kaplayacak şekilde kameranın ...

Watch this Scientific Journal Video about Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling at JoVE.com

Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling

This paper describes a protocol that assesses the changes of myofilament Ca2+ sensitivity during contraction in isolated cardiac myocytes from rat heart. Together with cardiac electrophysiology, systolic/diastolic cytosol Ca2+ levels and contraction/relaxation, this measurement is imperative in underpinning the mechanisms mediating cardiac excitation-contraction coupling in healthy and diseased hearts.

Myofilament Ca Değerlendirilmesi<sup&gt; 2+</sup&gt; Duyarlılık temelinde Kardiyak Uyarma-kasılma Kaplin

Heart failure and cardiac arrhythmias are the leading causes of mortality and morbidity worldwide. However, the mechanism of pathogenesis and myocardial ...

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Biology

Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling

9.4K Görüntüleme.  Seoul National University College of Medicine. This paper describes a protocol that assesses the changes of myofilament Ca2+ sensitivity during contraction in isolated cardiac myocytes from rat heart. Together with cardiac electrophysiology, systolic/diastolic cytosol Ca2+ levels and contraction/relaxation, this measurement is imperative in underpinning the mechanisms mediating cardiac excitation-contraction coupling in healthy and diseased hearts. 

Video: Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling

Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum (SR) serves as a Ca2+ reservoir for contraction, which reuptakes intracellular Ca2+ during relaxation. The SR Ca2+ reserve available for beats is determinate for cardiac contractibility, and the removal of intracellular Ca2+ is critical for cardiac diastolic function. Under some pathophysiological conditions, such as diabetes and heart failure, impaired calcium clearance and SR Ca2+ store in cardiomyocytes may be involved in the progress of cardiac dysfunction. Here, we describe a protocol to evaluate SRCa2+ reserve and diastolic Ca2+ removal. Briefly, a single cardiomyocyte was enzymatically isolated, and the intracellular Ca2+ fluorescence indicated by Fura-2 was recorded by a calcium imaging system. To employ caffeine for inducing total SR Ca2+ release, we preset an automatic perfusion switch program by interlinking the stimulation system and the perfusion system. Then, the mono-exponential curve fitting was used for analyzing decay time constants of calcium transients and caffeine-induced calcium pulses. Accordingly, the contribution of the SR Ca2+-ATPase (SERCA) and Na+-Ca2+ exchanger (NCX) to diastolic calcium removal was evaluated.

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Here, we describe a protocol to evaluate sarcoplasmic reticulum Ca2+ reserve and diastolic calcium removal function in cardiomyocytes by a calcium imaging system.

Sarcoplasmic retikulum kalsiyum koruma ve hücre içi diyastolik kalsiyum kaldırma izole Ventriküler Cardiomyocytes olarak değerlendirilmesi

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum ...

Tıp

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and geometric&#160;deformation. Our understanding of the internal relationships between these events has been limited because we can neither &#39;see&#39; inside the muscle nor precisely track the spatio-temporal nature of excitation-contraction dynamics. To resolve these problems, we have constructed a device that combines a suite of imaging modalities. Specifically, it integrates a brightfield microscope to measure local changes of sarcomere length and tissue strain, a fluorescence microscope to visualize the Ca2+ transient, and an optical coherence tomograph to capture the tissue&#39;s geometric&#160;changes throughout the time-course of a cardiac cycle. We present here the imaging infrastructure and associated data collection framework. Data are collected from isolated rod-like tissue structures known as trabeculae carneae. In our instrument, a pair of position-controlled platinum hooks hold each end of an ex vivo muscle sample while it is continuously superfused with nutrient-rich saline solution. The hooks are under independent control, permitting real-time control of muscle length and force. Lengthwise translation enables the piecewise scanning of the sample, overcoming limitations associated with the relative size of the microscope imaging window (540 &#181;m by 540 &#181;m) and the length of a typical trabecula (&#62;2000 &#181;m). Platinum electrodes at either end of the muscle chamber stimulate the trabecula at a user-defined rate. We exploit the stimulation signal as a trigger for synchronizing the data from each imaging window to reconstruct the entire sample twitching under steady-state conditions. Applying image-processing techniques to these brightfield imaging data provides tissue displacement and sarcomere length maps. Such a collection of data, when incorporated into an experiment-modeling pipeline, will provide a deeper understanding of muscle contractile homogeneity and heterogeneity in physiology and pathophysiology.

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

This protocol presents a collection of sarcomere, calcium, and macroscopic geometric&#160;data from an actively contracting cardiac trabecula ex vivo. These simultaneous measurements are made possible by the integration of three imaging modalities.

Kardiyak Trabeculae Ex Vivo'nun Eşzamanlı Brightfield, Floresan ve Optik Tutarlılık Tomografik Görüntülemesi

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and ...

Biyomühendislik

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

İzole İskelet Kaslar Kasılma, çabuk yorulma ve Alternans Ex Vivo Değerlendirilmesi

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

Biyoloji

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Kalsiyum değerlendirilmesi Bozulmamış İskelet kas liflerinin içinde Sparks

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

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 ...

Electromechanical Assessment of Optogenetically Modulated Cardiomyocyte Activity

Over the past two decades, optogenetic tools have been established as potent means to modulate cell-type specific activity in excitable tissues, including the heart. While Channelrhodopsin-2 (ChR2) is a common tool to depolarize the membrane potential in cardiomyocytes (CM), potentially eliciting action potentials (AP), an effective tool for reliable silencing of CM activity has been missing. It has been suggested to use anion channelrhodopsins (ACR) for optogenetic inhibition. Here, we describe a protocol to assess the effects of activating the natural ACR GtACR1 from Guillardia theta in cultured rabbit CM. Primary readouts are electrophysiological patch-clamp recordings and optical tracking of CM contractions, both performed while applying different patterns of light stimulation. The protocol includes CM isolation from rabbit heart, seeding and culturing of the cells for up to 4 days, transduction via adenovirus coding for the light-gated chloride channel, preparation of patch-clamp and carbon fiber setups, data collection and analysis. Using the patch-clamp technique in whole-cell configuration allows one to record light-activated currents (in voltage-clamp mode, V-clamp) and AP (current-clamp mode, I-clamp) in real time. In addition to patch-clamp experiments, we conduct contractility measurements for functional assessment of CM activity without disturbing the intracellular milieu. To do so, cells are mechanically preloaded using carbon fibers and contractions are recorded by tracking changes in sarcomere length and carbon fiber distance. Data analysis includes assessment of AP duration from I-clamp recordings, peak currents from V-clamp recordings and force calculation from carbon fiber measurements. The described protocol can be applied to the testing of biophysical effects of different optogenetic actuators on CM activity, a prerequisite for the development of a mechanistic understanding of optogenetic experiments in cardiac tissue and whole hearts.

We present a protocol for evaluating the electromechanical effects of GtACR1 activation in rabbit cardiomyocytes. We provide detailed information on cell isolation, culturing and adenoviral transduction, and on functional experiments with the patch-clamp and carbon-fiber techniques.

Optogenetik Modüle Kardiyomiyosit Aktivitesinin Elektromekanik Değerlendirilmesi

Over the past two decades, optogenetic tools have been established as potent means to modulate cell-type specific activity in excitable tissues, including ...

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics.

Farelerde Kardiyak inotropiyi Soruşturma İzole papiller Kaslar kontraktilite Ölçümleri

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a significant role in certain types of arrhythmia. Because arrhythmias are spatially distinct, emergent phenomena, they must be investigated at the tissue level. However, methods for directly probing SR Ca2+ in the intact heart remain limited. This article describes the protocol for dual optical mapping of transmembrane potential (Vm) and free intra-SR [Ca2+] ([Ca2+]SR) in the Langendorff-perfused rabbit heart. This approach takes advantage of the low-affinity Ca2+ indicator Fluo-5N, which has minimal fluorescence in the cytosol where intracellular [Ca2+] ([Ca2+]i) is relatively low but exhibits significant fluorescence in the SR lumen where [Ca2+]SR is in the millimolar range. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the distinct advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+ and the role of SR Ca2+ in arrhythmogenic phenomena.

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

This article describes the detailed protocol and equipment necessary for dual optical mapping of transmembrane potential (Vm) and free intra-sarcoplasmic reticulum (SR) Ca2+ in the Langendorff-perfused rabbit heart. This method allows for direct observation and quantification of Vm and SR Ca2+ dynamics in the intact heart.

Intra-Sarkoplazmik Retikulum Ca Optik Haritalama<sup&gt; 2+</sup&gt; Ve Langendorff-perfüze Tavşan Heart Transmembran Potansiyeli

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a ...

Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes

Contractile dysfunction and Ca2+ transients are often analyzed at the cellular level as part of a comprehensive assessment of cardiac-induced injury and/or remodeling. One approach for assessing these functional alterations utilizes unloaded shortening and Ca2+ transient analyses in primary adult cardiac myocytes. For this approach, adult myocytes are isolated by collagenase digestion, made Ca2+ tolerant, and then adhered to laminin-coated coverslips, followed by electrical pacing in serum-free media. The general protocol utilizes adult rat cardiac myocytes but can be readily adjusted for primary myocytes from other species. Functional alterations in myocytes from injured hearts can be compared to sham myocytes and/or to in vitro therapeutic treatments. The methodology includes the essential elements needed for myocyte pacing, along with the cell chamber and platform components. The detailed protocol for this approach incorporates the steps for measuring unloaded shortening by sarcomere length detection and cellular Ca2+ transients measured with the ratiometric indicator Fura-2 AM, as well as for raw data analysis.

Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes

A set of protocols are presented that describe the measurement of contractile function via sarcomere length detection along with calcium (Ca2+) transient measurement in isolated rat myocytes. The application of this approach for studies in animal models of heart failure is also included.

Kemirgen Miyositlerde Kardiyak Kasılma Disfonksiyonu ve Ca2+ Geçicilerinin Analizi

Contractile dysfunction and Ca2+ transients are often analyzed at the cellular level as part of a comprehensive assessment of cardiac-induced injury ...

Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility. If structural damage is present in the myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-P&#233;rot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.

Presented here is a protocol to assess the contractile properties of striated muscle myofibrils with nano-Newton resolution. The protocol employs a setup with an interferometry-based, optical force probe. This setup generates data with a high signal-to-noise ratio and enables the assessment of the contractile kinetics of myofibrils.

Myofilament Ca Değerlendirilmesi

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