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

동물과 관련된 모든 절차는 캘리포니아 데이비스 대학의 동물 관리 및 사용위원회의 승인을, 건강의 국립 연구소에 의해 게시 된 실험 동물의 관리 및 사용에 대한 가이드를 부착했다. 1. 준비 <ol><li> 4 ° C에서 사전 및 저장소에 수정 된 타이로드의 솔루션의 두 집중 (25X) 주식을 준비한다 : (1) 주식 I (MM에서 : 염화나트륨 3205, 염화칼슘 2 32.5, KCl을 117.5,의 NaH 2 PO 4 29.75,의 MgCl 2 26.25) 및 (2) (MM의 :의 NaHCO3 500) 주식 II. <ol><li> (DI)을 염화나트륨 128.2, CaCl2를 1.3의 KCl 4.7의 MgCl 2 1.05의 NaH 2 PO 4 1.19의 NaHCO3 (20) 및 탈 이온수 1840 ㎖에 결합하여 11.1 글루코스 : 갓 (㎜)에하기 조성의 2 L 타이로드의 용액을 제조 물 80 ml의 스톡 I, II의 스톡 80 ㎖, 및 글루코스 4g. 직접 주식 나는 그리고 스톡 II를 함께 사용하지 마십시오 추가1,840 ml의 DI에 m은 침전을 방지 할 수 있습니다. </li></ol></li><li> 사전에 10 mg을 무수 디 메틸 설폭 사이드 (DMSO)에 / ㎖에서 여기 수축 uncoupler의 blebbistatin의 주식 솔루션을 준비하고 4 ℃에서 원액을 저장합니다. </li><li> 형광 염료의 스톡 용액을 제조한다 : (1) 전압에 민감한 염료 RH237 (DMSO에 1 ㎎ / ㎖), (2) 낮은 친 칼슘 지시등 FLUO-5N AM 제조자의 권장 사항에 따라 (DMSO 2 ㎎ / ㎖)를. 적재 능력의 후속 손실 분해를 방지하기 위해 즉시 준비를 사용합니다. </li><li> 프라임 산소화 함께 재순환 랑겐 돌프 관류 시스템 (95 % O 2, 5 % CO 2)의 타이로드 용액. 7.4 ± 0.05의 타이로드 용액의 pH를 유지하기 위해, O2 / CO (2)의 흐름을 조정한다. 지속적으로 관류를 필터링 : 인라인 나일론 짠 그물 필터 (11 μm의 기공 크기)를 사용합니다. <ol><li> 이후 loadi으로 실온에서 국무 관류 시스템,FLUO-5N 오전 NG 실온에서 수행됩니다. FLUO-5N 오전 로딩, 순환 관류의 작은 볼륨 (~ 150 ㎖)를 사용합니다. 염료로드 한 후, 관류의 더 큰 볼륨 사용 (1-2 패, 그림 1C 참조). </li></ol></li><li> 데이터 수집 시스템의 전원을 켜고 심전도 및 관류 압력을 지속적으로 모니터링 준비합니다. 압력 모니터링 시스템을 준비합니다 관류 라인에 압력 변환기를 연결하고 transbridge 앰프 관류 압력을 모니터링 할 수 있습니다. 심장이 관류 시스템에 연결되지 않은 경우 0 mmHg로 기준선 관류 압력을 조정합니다. </li><li> V 분 및 SR 칼슘 신호 사이의 공간 정렬을 보장하기 위해 광학 매핑 카메라를 맞 춥니 다. <ol><li> 첫째, 통치자 또는 관류 접시에 텍스트 개체를 배치하여 카메라 초점을 맞 춥니 다. 라이브 뷰 모드로 매핑 카메라를 켜고 텍스트가 선명하고 선명해질 때까지 초점을 조절합니다. 각각의 카메라로부터 하나의 프레임 화상을 취득. </li><li> 오버레이두 이미지가 볼 수 있도록 이러한 이미지 및 상단 이미지의 투명도를 조정합니다. 이미지의 텍스트가 정확히 겹쳐한다. 완벽하게 정렬되지 않은 경우 두 이미지가 정렬 될 때까지, 다이크로 익 미러 또는 각 카메라의 위치의 각도를 조정한다. </li></ol></li></ol> 토끼 심장의 2. 수확, 관류, 그리고 염료 로딩 <ol><li> 승인 된 토끼 제한 수단의 토끼를 고정합니다. 깊이 펜 토바 비탈 나트륨 (50 mg / kg) 및 헤파린 (1000 IU)의 정맥 내 주사 (귀 가장자리 정맥)을 통해 마취. <ol><li> 토끼가 표시 침해 반사 신경이 부족하면, 중간 선 피부 절개는 흉골과 갈비뼈를 노출 할 수 있습니다. 흉골에 xyphoid에서 무딘 팁 수술 가위로 흉골을 통해 잘라. 흉골을 여는 동안 심장이 손상되지 않도록주의하십시오. 마음을 노출 갈비뼈를 확산. </li><li> 빠르게 빠르게 모든 선박과 결합 조직을 절단하여 전체 심장 - 폐 블록은 절제. 최단 당일tely 얼음처럼 차가운 타이로드의 용액에 심장 - 폐 블록을 배치합니다. </li></ol></li><li> 역 행성 관류의 대동맥을 찾습니다. 대동맥 궁의 세 가지에 바로 인접 상행 대동맥을 잘라. 관류 시스템에 연결 8 G 캐 뉼러에 대동맥 Cannulate. 캐 뉼러에 대동맥을 확보하기 위해 USP 0 실크 봉합사의 조각을 사용합니다. </li><li> 조심스럽게 마음에서 기관, 폐 및 심 외막 지방을 해부하다. 날카로운 집게 및 해부 가위, 승모판의 위치를​​주의 깊게 손상 또는 좌심실에서 솔루션 혼잡을 방지하기 위해 하나의 전단지를 제거합니다. </li><li> 이미징이 위를 향하도록 마음의 전방 표면과 수평으로 유리 외피 관류 실에 마음을 담근다. 매핑 (그림 1D) 동안 마음의 움직임을 방지하기 위해 접시의 실리콘 바닥에 U 자형 핀 실 가드의 조각에 캐 뉼러를 고정합니다. 원하는 경우, STA에 대한 심장의 정점에 작은 곤충 핀을 삽입bilization. </li><li> 위치 심전도 (ECG) 심장의 오른쪽과 왼쪽에 목욕 전극. 완전히 욕 전극 잠수함 그들이 리드 I 구성으로 부피가 유사한 실시 ECG 제공 심장 표면과 접촉하지 않는 보장. 보통의 리드 나는 심전도 형태를 확인합니다. 70 mmHg로 - 대동맥 압력이 60으로 유지 될 수 있도록 - (35 ml / 분 25) 흐름을 조정합니다. </li><li> 실내 조명을 끄고 0.3 추가 - 관류 (- 20 μm의 최종 농도 10)에 blebbistatin 주식 솔루션 (단계 1.2)의 0.6 ml에. <ol><li> blebbistatin의 photoinactivati​​on을 방지하기 위해 실내 조명을 끄십시오. 필요한 경우, 소형 스포트 또는 전조등은 작업 조명을 제공하기 위해 사용될 수있다. 주 :이 Blebbistatin 미오신 ATP 아제 억제제 및 여진 수축 uncoupler이다. 심장 에너지 생산이 손상 될 수있다 - FLUO-5N 로딩 확장 (60 분), RT (저체온증) 조건을 요구하기 때문에 (2.10 단계 2.8 참조). 목erefore, 관류 종래에 blebbistatin 첨가 에너지 수요 (16)을 감소시키고 후속 광학 레코딩 17 동안 모션 아티팩트를 제거 할 수있다 로딩 염료. </li></ol></li><li> 심장의 수축이 중단되면 (10 - 대동맥 압력 기록에 대한 확인 15 분), 작은 볼륨 순환 관류 시스템 (단계 1.4.1 참조)로 전환합니다. </li><li> 준비하고 FLUO-5N 오전 로딩 솔루션을로드 : 0.25 ml의 따뜻한 20 % 플루로 닉 F127에 FLUO-5N 오전 원액 (1.3 단계)의 0.25 ML을 추가합니다. 0.5 ml의 혼합물에 따뜻한 타이로드의 솔루션을 추가 잘 혼합하고, 소량 순환 관류 시스템에 추가 할 수 있습니다. </li><li> 연속적으로 관류 압력 및 ECG를 모니터하고 필요하다면 흐름을 조절한다. 염료 로딩은 실온에서 약 1 시간 소요됩니다. 로딩 후 45 분, 37 ℃로 관류 시스템 및 관류를 따뜻하게 시작 순환 물 목욕을 켜 시작했다. </li><li> FLUO-5N 오전 로딩, SWI의 60 분 후큰 부피 재순환 관류 시스템에 관류 TCH (단계 1.4 참조). </li><li> 따뜻한의 타이로드 액 1ml에 전압 감응 염료 RH237 원액 (단계 1.3) 50 μL를 희석하고 캐뉼라에 주입구 근위로 (~ 5 분에 걸쳐)을 천천히 부가. </li></ol> 3. 광학 매핑 <ol><li> 염료로드의 마지막 순간 동안, 마음을 놓고 실험에 대한보기의 해당 필드를 보장하기 위해 광학 매핑 카메라 초점을 맞 춥니 다. </li><li> 원하는 경우, 페이싱에 대한 마음의 심​​ 외막 표면에 양극 페이싱 전극을 배치합니다. </li><li> 인해 관류의 재순환에 존재할 수있는 액체의 표면에 동 잡음을 줄이기 위해 관류 챔버의 표면에 플라스틱이나 유리 커버 슬립을 배치했다. 주 : 커버 슬립에 대한 대안이 깨끗한 플라스틱 50mm 페트리 접시 덮개를 사용할 때. </li><li> 균일 전자와 마음의 표면을 조명 빛 가이드를 초점xcitation 빛. 495 nm의 대역 통과 필터 (도 1a) - 청색 발광 다이오드 (LED) 광원을 사용하여 상기 (475)로 광을 필터링. </li><li> macroscope 두 상보성 금속 산화물 반도체 (CMOS) 광학 매핑 카메라와 방출되는 형광을 수집합니다. 545 nm에서 이색 거울 방출 된 빛을 분할합니다. <ol><li> 롱 패스는 700 nm에서, V의 m 신호를 포함하는, 긴 파장 부분을 필터링. 534 나노 미터 (도 1A) - 밴드 패스 (502)에서, SR 칼슘 신호를 포함하는, 단파장 잔기 필터. 1 kHz에서 - 0.5 광 데이터 수집의 빈도를 설정합니다. </li></ol></li><li> 각각의 광 기록, 처음으로, 원하는 페이싱 프로토콜을 시작 여기 광을 켜고 1 수집 - 데이터의 4 초. 필요한 경우, 페이징 프로토콜 및 데이터 획득을 동기화. 주 : 것은 불가능 FLUO-5N AM, 따라서 신호 대 잡음비 (SNR)를 재 로딩한다 decrea자체 SR에서 누출 염료에 의한 실험을하는 동안. 높은 SNR 값을 보장하기 위해 2 시간 - 1 실험 프로토콜을 제한합니다. </li><li> 실험 후, 탈 이온수로 다시 DI 물, 70 % 알코올 시약의 순차적 관류 시스템을 씻어. </li><li> 제조사의 프로토콜에 따라 상업적으로 이용 가능한 소프트웨어 패키지를 이용하여 공간 가우시안 필터 (반경 3 픽셀)를 각 데이터 세트를 필터링. </li><li> 필요한 경우, 공간적으로 V의 m 및 SR 칼슘 2 + 데이터 세트를 맞 춥니 다. 단계 1.6에서와 같이, 각각의 카메라에서 이미지를 중첩되는지 확인보기 (즉, 정맥, 페이싱 전극)의 분야에서 심장의 해부학 적 구조 (즉., 관상 동맥 혈관, 심방의 가장자리 또는 심실) 또는 기타 항목을 정확하게 중복. 그렇지 않은 경우, 데이터 세트의 공간 배치가 필요하다. </li><li> 화소 번호를 기록하게 정확한 오버랩까지 X 축 및 Y 방향이 달성에 상부 투명 이미지를 이동이미지는 각 방향으로 이동해야합니다. 상부 이미지 (V m 또는 SR 칼슘 하나)에 대응하는 전체 데이터 세트는 V의 m 및 SR 칼슘 2 데이터 세트 사이의 정확한 정렬을 보장하기 위해, 각 방향의 화소 수만큼 시프트 결정되어야한다. </li></ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Luminal+Ca(2+)+content+regulates+intracellular+Ca(2+)+release+in+subepicardial+myocytes+of+intact+beating+mouse+hearts:+effect+of+exogenous+buffers.">Luminal Ca(2+) content regulates intracellular Ca(2+) release in subepicardial myocytes of intact beating mouse hearts: effect of exogenous buffers.</a> American journal of physiology. Heart and circulatory physiology. 298, H2138-H2153 (2010).</li><li>Picht, E., DeSantiago, J., Blatter, L. A., Bers, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+alternans+do+not+rely+on+diastolic+sarcoplasmic+reticulum+calcium+content+fluctuations.">Cardiac alternans do not rely on diastolic sarcoplasmic reticulum calcium content fluctuations.</a> Circulation research. 99, 740-748 (2006).</li><li>Shkryl, V. M., Maxwell, J. T., Domeier, T. L., Blatter, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refractoriness+of+sarcoplasmic+reticulum+Ca2++release+determines+Ca2++alternans+in+atrial+myocytes.+American+journal+of+physiology.">Refractoriness of sarcoplasmic reticulum Ca2+ release determines Ca2+ alternans in atrial myocytes. 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.

성공적인 FLUO-5N 염료 로딩 키 염료 대량의 로딩 시간 (1 시간)의 길이에 대한 필요없이 높은 FLUO-5N 농도 허용 소량 재순환 관류 셋업하고, 로딩을 수행 실온에서. 로딩 빠르게 생리 온도, 셀룰러 절단 효소 활성 염료 세포질에서 염료 분자를 포착하고이를 SR 세포막을 통과 할 수없는, 세포막을 통과 -AM 태그에서 수행되는 경우. RT에서, 그러나, 효소 활성은 둔화되고 -AM 태그 전에 세포막과 SR 막 모두...

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.

도 1a는 이중 V의 m 및 SR 칼슘 매핑 광학 구성의 개략도를 나타낸다. 이 설정으로, V의 m 및 SR 칼슘 신호 (그림 1B)의 전체 스펙트럼 분리가있다. FLUO-5N 염료 로딩에 사용되는 이중 루프 관류 시스템의 도면은도 1c에 도시된다.도 1d는 관류 접시 심장의 가로 방향을 나타낸다. 대표적인 V의 M과 심장의 심 외막 표면에 다른 ?...

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

이 문서에서는 랑겐 돌프 - 관류 토끼 심장의 상세한 프로토콜과 횡단 전위 (V 분)의 이중 광학 매핑 및 무료 내 근질 세망 (SR)에 필요한 장비 칼슘을 설명합니다. 이 방법은 그대로 심장에 직접 관찰 및 V (M)의 정량 및 SR 칼슘 역학 수 있습니다.

인트라 근질 그물 자리 칼슘의 광학 매핑<sup&gt; 2 +</sup&gt;와 랑겐 돌프 - 관류 토끼 심장에 막 횡단 잠재력

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 조회수.  University of California Davis.  이 문서에서는 랑겐 돌프 - 관류 토끼 심장의 상세한 프로토콜과 횡단 전위 (V 분)의 이중 광학 매핑 및 무료 내 근질 세망 (SR)에 필요한 장비 칼슘을 설명합니다. 이 방법은 그대로 심장에 직접 관찰 및 V (M)의 정량 및 SR 칼슘 역학 수 있습니다. 

비디오: 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.

현미경의 올바른 관리 및 청소

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

연구

생물학

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).

Langendorff - perfused 랫 하트의 광학 매핑

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.

에 잔 심장을 떠올리 Drosophila</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.

작은 하트의 세미 - 자동 광학 도면 분석

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.

의 전자 현미경을위한 배아의 준비 Drosophila 태아의 심장 관

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.

Ionising 방사선에 대한 응답으로 γH2AX Foci의 부량

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.

이온화 방사선 다음 γH2AX의 공간적 분포 평가

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.

Clonogenic 분석 : 자기편 셀

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.

배양 된 HL-1 심방 근육 세포 단층의 전압 및 칼슘 듀얼 채널 광학 매핑

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.