This work was supported in part by the US National Institutes of Health (R01 HL 111600) and the American Heart Association (12SDG9010015).

وقد وافق جميع الإجراءات التي تنطوي على الحيوانات من قبل لجنة رعاية واستخدام الحيوان من جامعة كاليفورنيا في ديفيز، والالتزام بها دليل لرعاية واستخدام الحيوانات المختبرية التي نشرتها المعاهد الوطنية للصحة. 1. إعداد <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد اثنين تتركز (25X) الأسهم المعدلة حل تايرود مسبقا وتخزينها في 4 ° C: (1) ألبوم I (في ملي: كلوريد الصوديوم 3205، CaCl 2 32.5، بوكل 117.5، ناه 2 PO 4 29.75، MgCl 2 26.25) و (2) سهم II (في ملي: NaHCO 3 500). <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> طازجة إعداد الحل 2 L تايرود من التشكيل التالي (مم): كلوريد الصوديوم 128.2، CaCl 2 1.3، بوكل 4.7، MgCl 2 1.05 ناه 2 PO 4 1.19، NaHCO 3 20 والجلوكوز 11.1 عن طريق الجمع بين 1840 مل من منزوع الأيونات (DI) الماء، 80 مل الأسهم I، 80 مل من الأسهم II، و 4 غرام من الجلوكوز. لا تخلط مباشرة المالية الأول والمالية II؛ إضافةم إلى 1840 مل DI لتجنب الأمطار. </li></ol></li><li style=";text-align:right;direction:rtl"> إعداد محلول المخزون من الإثارة، تقلص uncoupler blebbistatin في 10 ملغ / مل في اللامائية دى methylsulfoxide (DMSO) مقدما وتخزين الحل السهم عند 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> إعداد الحلول الأسهم من الأصباغ الفلورية: (1) RH237 الجهد تراعي صبغ (1 ملغ / مل في DMSO) و (2) انخفاض مؤشر تقارب الكالسيوم فلوو-5N AM (2 ملغ / مل في DMSO) وفقا لتوصيات الشركة الصانعة. استخدام الاستعدادات على الفور لتجنب التحلل مع الخسارة اللاحقة من قدرة التحميل. </li><li style=";text-align:right;direction:rtl"> رئيس إعادة تدوير نظام نضح Langendorff مع الاوكسيجين (95٪ O 2، 5٪ CO 2) حل تايرود. ضبط تدفق O 2 / CO 2 للحفاظ على درجة الحموضة من الحل تايرود بنسبة 7.4 ± 0.05. استخدام صافي مرشح في خط النايلون المنسوجة (حجم المسام: 11 ميكرون) لتصفية باستمرار الإرواء. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> رئيس نظام نضح في RT، كما loadi اللاحقةيتم تنفيذ نانوغرام من فلوو-5N AM في RT. لفلوو-5N AM التحميل واستخدام كمية صغيرة من إعادة تدوير الإرواء (~ 150 مل). بعد صبغ التحميل واستخدام كميات أكبر من سائل الإرواء (1-2 L، انظر الشكل 1C). </li></ol></li><li style=";text-align:right;direction:rtl"> تشغيل نظام الحصول على البيانات وتحضير لأعمال الرصد المستمر لECG ونضح الضغط. إعداد نظام الضغط رصد: قم بتوصيل محول الضغط إلى خط نضح ورصد ضغط التروية مع مكبر للصوت transbridge. ضبط ضغط التروية الأساسي ل0 مم زئبق عندما لا يرد القلب إلى نظام الارواء. </li><li style=";text-align:right;direction:rtl"> محاذاة كاميرات رسم الخرائط البصرية لضمان المواءمة المكانية بين م V وريال كا 2+ الإشارات. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> أولا، تركيز الكاميرات عن طريق وضع مسطرة أو كائن مع النص في طبق الارواء. تحويل كاميرات رسم الخرائط في وضع العرض الحي وضبط التركيز حتى النص هو هش واضحة. الحصول على صورة إطار واحد من كل كاميرا. </li><li style=";text-align:right;direction:rtl"> غطاءهذه الصور وضبط شفافية الصورة العليا بحيث أن كلا من صور مرئية. النص في الصور يجب أن تتداخل تماما. إن لم يكن المنحازة تماما، وضبط زاوية المرآة مزدوج اللون أو موقف كل الكاميرا حتى يتم محاذاة الصورتين. </li></ol></li></ol> 2. الحصاد، والإرواء، وصبغ تحميل الأرنب القلب <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تأمين أرنب في رادع أرنب المعتمدة. تخدير عميق عن طريق الحقن في الوريد (الوريد الأذن هامشية) من بنتوباربيتال الصوديوم (50 ملغ / كلغ) والهيبارين (1000 IU). <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> عندما تفتقر يعرض أرنب من ردود الفعل مسبب للألم، وجعل شق الجلد خط الوسط لفضح القص والضلوع. قطع طريق القص مع صريحا معلومات سرية مقص جراحي من سيفي الشكل إلى المقبض. الحرص على عدم تلف القلب أثناء فتح القص. نشر الأضلاع لفضح القلب. </li><li style=";text-align:right;direction:rtl"> المكوس بسرعة كامل كتلة القلب والرئة عن طريق خفض سرعة جميع السفن والنسيج الضام. Immediaوضع tely كتلة القلب والرئة إلى حل المثلج تايرود. </li></ol></li><li style=";text-align:right;direction:rtl"> تحديد الشريان الأورطي لنضح الوراء. قطع الشريان الأبهر الصاعد للتو الأقرب إلى الفروع الثلاثة للقوس الأبهر. يقني؛ يدخل القنية الشريان الأورطي إلى قنية 8 G متصلا نظام الارواء. استخدام قطعة من USP 0 خياطة الحرير لتأمين الشريان الأورطي على قنية. </li><li style=";text-align:right;direction:rtl"> تشريح بعناية القصبة الهوائية والرئتين، والدهون النخابية من القلب. مع ملقط حادة ومقص تشريح، موقع الصمام التاجي وبعناية ضرر أو إزالة النشرة واحدة لمنع الازدحام الحل في البطين الأيسر. </li><li style=";text-align:right;direction:rtl"> يغرق القلب في غرفة نضح-تغلف الزجاج أفقيا مع السطح الأمامي للقلب مواجهة للتصوير. تأمين قنية إلى قطعة من Sylgard مع دبابيس على شكل حرف U على الجزء السفلي من السيليكون الطبق لمنع حركة القلب أثناء رسم الخرائط (1D الشكل). إذا رغبت في ذلك، اضافة الى وجود دبوس حشرة صغيرة في قمة القلب لستاbilization. </li><li style=";text-align:right;direction:rtl"> موقف الكهربائي (ECG) الأقطاب الكهربائية في الحمام على الجانب الأيمن والأيسر من القلب. يغرق تماما الأقطاب في الحمام والتأكد من أنهم ليسوا على اتصال مع سطح القلب لتوفير ECG-أجرت حجم مماثل لتكوين I الرصاص. التحقق من الرصاص العادي I ECG التشكل. ضبط تدفق (25-35 مل / دقيقة) للتأكد من أن ضغط الأبهر هو الحفاظ على 60-70 مم زئبق. </li><li style=";text-align:right;direction:rtl"> إطفاء الأنوار الغرفة وإضافة 0،3-0،6 مل من محلول المخزون blebbistatin (الخطوة 1.2) إلى الإرواء (تركيز النهائي 10-20 ميكرومتر). <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إطفاء الأنوار الغرفة لتجنب تعطيل ضوئي من blebbistatin. إذا لزم الأمر، الضوء صغير أو كشافات يمكن أن تستخدم لتوفير الإضاءة المهمة. ملاحظة: Blebbistatin هو الميوسين أتباز المانع وuncoupler الإثارة، تقلص. لأن فلوو-5N التحميل يتطلب طويلة (60 دقيقة) RT (انخفاض حرارة الجسم) شروط (انظر الخطوات 2،8 حتي 2،10)، قد يكون خطر إنتاج الطاقة القلبية. عشرerefore، إضافة blebbistatin في الإرواء قبل أن تصبغ التحميل قد يقلل من الطلب على الطاقة 16 و القضاء على الحركة الفنية خلال التسجيلات البصرية اللاحقة 17. </li></ol></li><li style=";text-align:right;direction:rtl"> عندما توقف انقباض القلب (10-15 دقيقة، والتحقق منها على تسجيل ضغط الأبهر)، والتحول إلى نظام إعادة تدوير نضح صغيرة الحجم (راجع الخطوة 1.4.1). </li><li style=";text-align:right;direction:rtl"> إعداد وتحميل فلوو-5N AM تحميل الحل: إضافة 0.25 مل من فلوو-5N AM الأسهم الحل (الخطوة 1.3) إلى 0.25 مل دافئ 20٪ F127 Pluronic. إضافة 0.5 مل من محلول دافئ تايرود إلى الخليط، وتخلط جيدا، وإضافة إلى نظام إعادة تدوير نضح صغيرة الحجم. </li><li style=";text-align:right;direction:rtl"> مراقبة مستمرة ضغط التروية وECG وضبط تدفق إذا لزم الأمر. صبغ التحميل يستغرق حوالي 1 ساعة على RT. 45 دقيقة بعد تحميل بدأت، بدوره على حمام ماء المتداولة لبدء ارتفاع درجة حرارة نظام نضح وسائل الإرواء إلى 37 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> بعد 60 دقيقة من فلوو-5N AM التحميل، مفاتتك نضح في وحدة تخزين النظام إعادة تدوير نضح أكبر (راجع الخطوة 1.4). </li><li style=";text-align:right;direction:rtl"> تمييع 50 ميكرولتر من الجهد حساسة محلول المخزون صبغ RH237 (الخطوة 1.3) في 1 مل من محلول دافئ تايرود وإضافة ببطء (أكثر من ~ 5 دقائق) إلى الأقرب ميناء حقن للقنية. </li></ol> 3. الخرائط الضوئية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> خلال اللحظات الأخيرة من صبغ تحميل، ضع قلب وتركيز الكاميرات رسم الخرائط الضوئية لضمان الحقل المناسب النظر للتجربة. </li><li style=";text-align:right;direction:rtl"> إذا رغبت في ذلك، ضع سرعة الكهربائي بين القطبين على سطح النخابية من القلب للسرعة. </li><li style=";text-align:right;direction:rtl"> وضع البلاستيك أو الزجاج غطاء الانزلاق على سطح غرفة نضح للحد من حركة القطع الأثرية على سطح السائل التي قد تكون موجودة بسبب إعادة تدوير سائل الإرواء. ملاحظة: كبديل لزلة غطاء، واستخدام البلاستيك 50 ملم بيتري غطاء طبق نظيف. </li><li style=";text-align:right;direction:rtl"> تركيز أدلة الخفيفة لإلقاء الضوء بشكل موحد على سطح القلب مع البريدضوء xcitation. استخدام الضوء الأزرق الصمام الثنائي الباعث لل(LED) مصادر الضوء وكذلك تصفية ضوء مع 475-495 نانومتر مرشح ممر الموجة (الشكل 1A). </li><li style=";text-align:right;direction:rtl"> جمع مضان المنبعثة مع macroscope واثنين تكميلية المعادن أكسيد أشباه الموصلات (CMOS) كاميرات رسم الخرائط البصرية. تقسيم الضوء المنبعث مع مرآة مزدوج اللون في 545 نانومتر. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تمرير طويل تصفية أطول الطول الموجي شاردة، التي تحتوي على V متر إشارة، في 700 نانومتر. الفرقة تمرير تصفية أقصر طول موجي شاردة، التي تحتوي على الكالسيوم ريال 2+ إشارة، 502-534 نانومتر (الشكل 1A). ضبط تردد من الحصول على البيانات الضوئية ل0،5-1 كيلو هرتز. </li></ol></li><li style=";text-align:right;direction:rtl"> لكل تسجيل البصرية، والشروع في أول بروتوكول سرعة المطلوبة، بدوره على ضوء الإثارة، وجمع 1-4 ثانية من البيانات. إذا رغبت في ذلك، وتزامن اكتساب بروتوكولات سرعة والبيانات. ملاحظة: ليس من الممكن لإعادة تحميل وفلوو-5N AM، وبالتالي فإن نسبة الإشارة إلى الضوضاء (SNR) سوف decreaحد ذاتها في كافة مراحل التجربة بسبب صبغ تسرب من SR. لحد بروتوكول تجريبي ل1-2 ساعة لضمان القيم SNR عالية. </li><li style=";text-align:right;direction:rtl"> بعد التجربة، وغسل نظام نضح في سلسلة من المياه DI و 70٪ كاشف الكحول، ومرة ​​أخرى بالماء DI. </li><li style=";text-align:right;direction:rtl"> تصفية كل مجموعة بيانات مع فلتر التمويه المكاني (دائرة نصف قطرها 3 بكسل) باستخدام حزم البرامج المتاحة تجاريا وفقا لبروتوكول الشركة المصنعة. </li><li style=";text-align:right;direction:rtl"> إذا لزم الأمر، محاذاة مكانيا م V ومجموعات البيانات الكالسيوم 2+ ريال. كما في الخطوة 1.6، تراكب الصور من كل كاميرا والتحقق من أن الهياكل التشريحية للقلب (أي.، الأوعية الدموية التاجية، وحواف من الأذينين أو البطينين) أو العناصر الأخرى في مجال الرؤية (أي، قنية، سرعة القطب) بالضبط التداخل. إن لم يكن، والمحاذاة المكانية من قواعد البيانات غير الضرورية. </li><li style=";text-align:right;direction:rtl"> تحويل أعلى صورة شفافة في يتحقق X- و ص الاتجاه حتى التداخل المحدد، مما يجعل علما أن عدد بكسليجب أن تحول صورة في كل اتجاه. مجموعة البيانات بأكملها الموافق الصورة العليا (إما V م أو الكالسيوم ريال 2+) ثم يجب أن تحول من قبل عدد محدد من بكسل في كل اتجاه لضمان المواءمة الدقيقة بين م V وريال كا 2+ قواعد البيانات. </li></ol>

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American journal of physiology.</a> Heart and circulatory physiology. 302, H2310-H2320 (2012).</li><li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+mapping+of+sarcoplasmic+reticulum+Ca2++in+the+intact+heart:+ryanodine+receptor+refractoriness+during+alternans+and+fibrillation.">Optical mapping of sarcoplasmic reticulum Ca2+ in the intact heart: ryanodine receptor refractoriness during alternans and fibrillation.</a> Circulation research. 114, 1410-1421 (2014).</li><li>Wengrowski, A. M., Kuzmiak-Glancy, S., Jaimes, R., Kay, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NADH+Changes+During+Hypoxia,+Ischemia,+and+Increased+Work+Differ+Between+Isolated+Heart+Preparations.">NADH Changes During Hypoxia, Ischemia, and Increased Work Differ Between Isolated Heart Preparations.</a> American journal of physiology. Heart and circulatory physiology. , (2013).</li><li>Fedorov, V. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+blebbistatin+as+an+excitation-contraction+uncoupler+for+electrophysiologic+study+of+rat+and+rabbit+hearts.">Application of blebbistatin as an excitation-contraction uncoupler for electrophysiologic study of rat and rabbit hearts.</a> Heart rhythm : the official journal of the Heart Rhythm Society. 4, 619-626 (2007).</li><li>Myles, R. C., Wang, L., Kang, C., Bers, D. M., Ripplinger, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+beta-adrenergic+stimulation+overcomes+source-sink+mismatch+to+generate+focal+arrhythmia.">Local beta-adrenergic stimulation overcomes source-sink mismatch to generate focal arrhythmia.</a> Circulation research. 110, 1454-1464 (2012).</li><li>Marks, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+cycling+proteins+and+heart+failure:+mechanisms+and+therapeutics.">Calcium cycling proteins and heart failure: mechanisms and therapeutics.</a> The Journal of clinical investigation. 123, 46-52 (2013).</li><li>Cutler, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+sarcoplasmic+reticulum+Ca2++ATPase+2a+gene+delivery+to+restore+electrical+stability+in+the+failing+heart.">Targeted sarcoplasmic reticulum Ca2+ ATPase 2a gene delivery to restore electrical stability in the failing heart.</a> Circulation. 126, 2095-2104 (2012).</li></ol>

The authors declare that they have no competing financial interests.

مفاتيح النجاح فلوو-5N صبغ التحميل هي صغيرة الحجم الإعداد إعادة تدوير التروية، والذي يسمح لتركيز فلوو-5N عالية دون الحاجة إلى كميات كبيرة من الصبغة، وطول وقت التحميل (1 ساعة)، وأداء التحميل في RT. إذا تم تنفيذ التحميل في درجات الحرارة الفسيولوجية والنشاط الأنزيمي الخلوي ?...

Dual optical mapping of intracellular Ca2+ and transmembrane potential (Vm) in the intact Langendorff-perfused heart has become a mainstay of investigations in cardiac electrophysiology, including mechanisms of arrhythmia and excitation-contraction coupling1-4. This approach has provided unprecedented knowledge into normal and abnormal electrophysiology and, importantly, into the bidirectional relationship between Vm and intracellular Ca2+. However, optical mapping of intracellular Ca2+ with high-affinity fluorescent indicators (such as Rhod-2 and Fluo-4) only reports on bulk changes in intracellular Ca2+ and is unable to distinguish whether these changes are due to transmembrane Ca2+ flux, release and reuptake into intracellular stores, or in most instances, some combination of both. Furthermore, high-affinity Ca2+ indicators have slow on-off kinetics and may not accurately report rapid changes in Ca2+ concentration5.
Each action potential triggers a rise in intracellular Ca2+, known as the intracellular Ca2+ transient (CaT). In the mammalian heart, approximately 70 &#8211; 90% of the total CaT is due to release of Ca2+ from the sarcoplasmic reticulum (SR) via opening of ryanodine receptors (RyRs)6. Within the SR, approximately half of the total Ca2+ is bound to calsequestrin (CSQ) and other intra-SR buffers7, which play an important role in SR Ca2+ homeostasis8,9. The amount of free SR Ca2+ dictates the driving force for SR Ca2+ release as well as gating of RyR, and therefore has a significant impact on the intracellular CaT. Furthermore, alterations in SR Ca2+ release or reuptake can, in turn, impact Vm via the electrogenic Na+-Ca2+ exchange, which may have arrhythmogenic consequences. Therefore, in addition to the CaT, monitoring of free SR Ca2+ can provide important insights into contractile and electrophysiological dysfunction.
Over the past several years, investigators have made significant advances in the monitoring of SR Ca2+ in isolated cardiac myocytes and from a single location on the intact heart. One such method requires rapid pulses of caffeine to open RyRs and the SR Ca2+ content is then inferred or calculated from the immediate rise in intracellular Ca2+10. Another intriguing approach uses low-affinity Ca2+ indicators, such as Fluo-5N11 or Mag-Fluo412, which bind to free SR Ca2+. These indicators have dissociation constants (Kd) in the range of 10 &#8211; 400 &#956;M and therefore exhibit minimal fluorescence in the cytosol compared to the SR lumen, where the Ca2+ concentration ([Ca2+]SR) is in the millimolar range. Using low-affinity Ca2+ indicators, several aspects of SR Ca2+ cycling have been investigated at the level of the isolated myocyte, including fractional SR Ca2+ release and the mechanisms of Ca2+ alternans13,14. However, in order to fully understand the heterogeneous nature of SR Ca2+ cycling in the intact heart and the role of SR Ca2+ in spatially distinct arrhythmic phenomena, methods for imaging SR Ca2+ across the epicardial surface of the intact heart are required15.
This article describes methodology for dual optical mapping of free SR Ca2+ and Vm in the intact Langendorff-perfused rabbit heart with the low-affinity Ca2+ indicator Fluo-5N. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+.

<table border="0" cellpadding="0" cellspacing="0" width="956">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">NaCl</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">S271-1</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">CaCl2 (2H2O)</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">C79-500</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">KCl</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">S217-500</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">MgCl2 (6H2O)</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">M33-500</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">NaH2PO4 (H2O)</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">S369-500</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">NaHCO3</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">S233-3</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">D-Glucose</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">D16-1</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">95% O2 5% CO2</td>
			<td style="width:167px;">AirGas</td>
			<td style="width:151px;">carbogen</td>
			<td style="width:423px;">For oxygenation and pH of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Blebbistatin</td>
			<td style="width:167px;">Tocris Bioscience</td>
			<td style="width:151px;">1760</td>
			<td style="width:423px;">Excitation-contraction uncoupler</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">RH237</td>
			<td style="width:167px;">Biotium</td>
			<td style="width:151px;">61018</td>
			<td style="width:423px;">Voltage-sensitive dye</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Fluo-5N AM</td>
			<td style="width:167px;">Invitrogen</td>
			<td style="width:151px;">F-26915</td>
			<td style="width:423px;">Low-affinity Ca2+ indicator; Alternative: Invitrogen F-14204; Loading must be performed at room temperature</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Pluronic F127</td>
			<td style="width:167px;">Biotium</td>
			<td style="width:151px;">59004</td>
			<td style="width:423px;">For Ca2+ indicator loading; Warm until the solutiion is clear before use</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Dimethyl sulphoxide (DMSO)</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:151px;">D2650</td>
			<td style="width:423px;">For dissolving blebbistatin and dyes</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Filter</td>
			<td style="width:167px;">EMD Millipore</td>
			<td style="width:151px;">NY1104700</td>
			<td style="width:423px;">11um in-line filter</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Pressure Transducer</td>
			<td style="width:167px;">WPI</td>
			<td style="width:151px;">BLPR2</td>
			<td style="width:423px;">For measuring perfusion pressure</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Transbridge Transducer Amplifier</td>
			<td style="width:167px;">WPI</td>
			<td style="width:151px;">SYS-TBM4M</td>
			<td style="width:423px;">For transducing/amplifing pressure signal; PowerLab may also be used with appropriate BioAmp</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">PowerLab 26T</td>
			<td style="width:167px;">ADInstruments</td>
			<td style="width:151px;"></td>
			<td style="width:423px;">For continuous recording of pressure and ECG signals</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">THT Macroscope</td>
			<td style="width:167px;">SciMedia</td>
			<td style="width:151px;"></td>
			<td style="width:423px;">Macroscopic optical setup. Details: 0.63x objective (NA=0.31), 2x condensing objective, resultant field of view = 3.1x3.1 cm, depth of focus = ~1.5mm</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">MiCam Ultima-L CMOS</td>
			<td style="width:167px;">SciMedia</td>
			<td style="width:151px;"></td>
			<td style="width:423px;">Optical mapping cameras</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Precision LED Spot Light</td>
			<td style="width:167px;">Mightex</td>
			<td style="width:151px;">PLS-0470-030-15-S</td>
			<td style="width:423px;">LED light source</td>
		</tr>
	</tbody>
</table>

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.

ويبين الشكل 1A الرسم التخطيطي من التكوين البصري للV المزدوج م وريال كا 2+ رسم الخرائط. مع هذا الإعداد، هناك فصل الطيفي الكامل للمتر V وريال كا 2+ إشارات (الشكل 1B). ويوضح شكل توضيحي لنظام نضح المزدوج حلقة استخدامها لفلوو-5N...

Watch this Scientific Journal Video about Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart at JoVE.com

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

توضح هذه المقالة بروتوكول مفصلة والمعدات اللازمة لرسم الخرائط الضوئية المزدوجة من الغشاء المحتملين (V م) وخالية شبكية داخل الهيولى العضلية (SR) كا 2+ في قلب الأرنب Langendorff perfused. وتسمح هذه الطريقة للمراقبة المباشرة والكمي لV م وريال كا 2+ ديناميكية في قلب سليمة.

رسم الخرائط البصرية البينية العضلية شبكية كاليفورنيا<sup&gt; 2+</sup&gt; وإمكانية الغشاء في قلب الأرنب Langendorff perfused-

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

optical-mapping-intra-sarcoplasmic-reticulum-ca2-transmembrane

Research

JoVE Journal

Biology

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

9.1K Views.  University of California Davis.  توضح هذه المقالة بروتوكول مفصلة والمعدات اللازمة لرسم الخرائط الضوئية المزدوجة من الغشاء المحتملين (V م) وخالية شبكية داخل الهيولى العضلية (SR) كا 2+ في قلب الأرنب Langendorff perfused. وتسمح هذه الطريقة للمراقبة المباشرة والكمي لV م وري?...

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

Proper Care and Cleaning of the Microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

العناية والتنظيف السليم للمجهر

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

Optical Mapping of Langendorff-perfused Rat Hearts

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs[1, 2]. Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly[1]. The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods[1]. Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency[1, 3]. In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37&deg;C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS[4] (5 &mu;Mol/L) and briefly illuminated the preparation with 475&plusmn;15 nm excitation light. During a typical 2 second period of illumination, &gt;605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

رسم الخرائط البصرية القلوب الجرذ Langendorff - perfused

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

تصور القلب النابض في ذبابة الفاكهة</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

شبه الآلي التحليل البصري نبضات القلوب الصغيرة

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

إعداد الأجنة المجهري للإلكترون من ذبابة الفاكهة أنبوب القلب الجنينية

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

الكمي بؤر γH2AX في الاستجابة للإشعاع المؤين

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &amp;#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

تقييم التوزيع المكاني للγH2AX التالية الإشعاعات المؤينة

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

مولد الرمع الفحص : خلايا منتسب

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte monolayers. HL-1 cells have been widely used as a 2-Dimensional cellular model for studying diverse aspects of cardiac physiology. However, it has been a great challenge to optically map calcium (Ca) transients and action potentials simultaneously from the same field of view in a cultured HL-1 atrial cell monolayer. This is because special handling and care is required to prepare healthy cells that can be electrically captured and optically mapped. Therefore, we have developed an optimal working protocol for dual channel optical mapping. In this manuscript, we have described in detail how to perform the dual channel optical mapping experiment. This protocol is a useful tool to enhance the understanding of action potential propagation and Ca kinetics in arrhythmia development.

This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.

الجهد والكالسيوم مزدوجة القناة البصرية رسم خرائط مثقف HL-1 الرجفان العضلية واحد رقائق

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte ...

High-resolution Optical Mapping of the Mouse Sino-atrial Node

Sino-atrial node (SAN) dysfunctions and associated complications constitute important causes of morbidity in patients with cardiac diseases. The development of novel pharmacological therapies to cure these patients relies on the thorough understanding of both normal physiology and pathophysiology of the SAN. Among the studies of cardiac pacemaking, the mouse SAN is widely used due to its feasibility for modifications in the expression of different genes that encode SAN ion channels or calcium handling proteins. Emerging evidence from electrophysiological and histological studies has also proved the representativeness and similarity of the mouse SAN structure and functions to larger mammals, including the presence of specialized conduction pathways from the SAN to the atrium and a complex pacemakers' hierarchy within the SAN. Recently, the technique of optical mapping has greatly facilitated the exploration and investigation of the origin of excitation and conduction within and from the mouse SAN, which in turn has extended the understanding of the SAN and benefited clinical treatments of SAN dysfunction associated diseases. In this manuscript, we have described in detail how to perform the optical mapping of the mouse SAN from the intact, Langendorff-perfused heart and from the isolated atrial preparation. This protocol is a useful tool to enhance the understanding of mouse SAN physiology and pathophysiology.

Here, we present a protocol for optical mapping of electrical activity from the mouse right atrium and especially the sino-atrial node, at a high spatial and temporal resolution.