We are supported by the University of Wisconsin-Madison Medical School start-up (A.V.G.).

모든 실험은 실험 동물의 관리 및 사용을위한 건강 가이드의 국립 연구소 (NIH 출판. 번호 80-23)에 따라 실시 하였다. 이 연구에 사용 된 모든 방법과 프로토콜 NIH에 의해 출판 실험실 동물의 관리 및 사용에 대한 지침은 다음 위스콘신 동물 관리 및 사용 프로토콜위원회의 대학에 의해 승인 된 (발행 번호 85-23를, 1996 년 개정). 본 연구에 사용 된 모든 동물 실험 동물의 관리 및 사용에 대한 가이드를 준수 인도적인 치료를 받았다. 1. 심장 제거 및 랑겐 돌프 관류 <ol><li> 심장 차단, 수조와 물 재킷을 사용하여 37 ° C 따뜻한 타이로드 용액에 앞서. </li><li> 5 % 이소 플루 란 / 95 % O 2 마우스를 마취. </li><li> 고통 반사의 손실을 확인하여 마취의 적절한 수준을 확인합니다. </li><li> 개흉술을 수행합니다. 곡선 5.5 &quot;메이요 가위 5.5&quot;켈리 지혈 용을 사용하여 가슴을 열고CEPS는 흉부의 전면에 잘라 1cm를 확인합니다. <ol><li> 신속하게 (30 초 이내) 가슴에서 심장의 압축을 풉니 다. &quot;폐 조직을 잡아 4.3 사용하여 심낭과 함께 폐, 흉선, 심장을 잘라 곡선 아이리스 집게 &#39;아이리스 가위 (4)를 사용합니다. 직접 마음을 잡아하지 마십시오. </li><li> 산소 (95 % O 2 / 5 % CO 2)에 씻어, 일정한 온도 (36.8 ± 0.4 ° C)의 타이로드의 솔루션을 수정했습니다. (MM)에서 다음과 용액 조성물을 사용하여 128.2 염화나트륨 4.7의 KCl을 1.19의 NaH 2 PO 4, 1.05의 MgCl 2 1.3 CaCl2를 20.0의 NaHCO3 및 글루코오스 11.1 (pH를 7.35 ± 0.05). </li></ol></li><li> 동일한 솔루션에 목욕하는 동안, 폐, 흉선, 지방 조직을 식별합니다. 조심스럽게 마음을 해부하다. 3 &quot;Vannas - 튀빙겐 가위 4.3&quot;# 5 집게 곡선 사용합니다. </li><li> 그런 다음 대동맥을 확인하고 주문 제작 21 G 정맥에 그것을 cannulate. 이 곡선 4.3 &quot;#의 5B의 힘을 사용하여추신. </li><li> 삽관 한 후 동시에 관류 (~ 50 ml / 분의 유속으로 목욕) (~ 5 ㎖ / 분의 유량에서의 캐뉼라를 통해가 대동맥 압력에 기초하여 조정되어야하며, 아래 참조) superfuse 전체 실험을 통해 예열 여과 타이로드 용액으로 마음입니다. 관상 동맥의 폐색을 방지하기 위해 인 - 라인 11 ㎛의 나일론 필터를 통해 관류 액이 통과한다. </li><li> 관류 라인에 3 방향 스톱 콕 루어 잠금 장치를 통해 연결된 압력 변환기 (또는 압력 게이지)를 사용하여 동맥압을 모니터 (60) 및 80 mmHg의 사이에 유지한다. 이 범위 내에서 대동맥 압력을 유지하기 위해 필요한 경우 관류 속도를 조정합니다. </li></ol> 랑겐 돌프 - 관류 항목의 심장에서 SAN 2. 광학 매핑 <ol><li> 수단 <ol><li> 수평으로 중심을두고 P 0.1 mm 직경의 핀을 이용하여 상기 챔버의 실리콘 코팅 아래쪽 심실 에이펙스 핀하지 못하 스트림에 의한 실험 기간 동안 운동을. (12) </li><li> 폐정맥을 통해 좌심실 (LV)에 작은 (0.012 &quot;(내경) x 0.005&quot;) 실리콘 튜브를 삽입, 왼쪽 심방 (LA)과 승모판 막 (MV). 사운 딩 결합 조직에 실크 봉합사 (4-0)에 의해 튜브를 고정합니다. </li><li> 그 후방 측 (그림 1A, 왼쪽 패널)를 향하도록 마음을 놓습니다. </li><li> 0.1 mm 직경 minutien 핀을 이용하여 상기 챔버의 실리콘 바닥까지 RA 부속기 (RAA)의 가장자리에 핀. 적경 평면의 후방 표면을하고 카메라의 초점면에 위치 할 수 있도록하기 위해 자사의 레벨을 조정합니다. 이는 심방의 최대 표면적 광학 측정을 가능하게 할 것이다. </li><li> 실크 봉합사 (4-0)에 의해 올가미 우수 (SVC) 및 하부 (IVC) 대정맥, 스트레칭과 실리콘 코팅 챔버 (그림 3A 참조)의 바닥에 봉합사의 다른 쪽 끝을 고정. 봉합사는 차단하지 않습니다 확인보기의 광학 분야. </li><li> RAA의 가장자리에 맞춤 페이싱 전극을 배치합니다. 전극하려면 사용 실리콘 0.5 mm 전극 간 거리, 0.25 mm 직경은 와이어를 코팅하고 조율 끝에 1mm의 길이에 대한 실리콘의 결여. 그런 다음 두 개의 ECG 12mm 바늘 (29 게이지) 좌우 심실의 기지 근처 모노 폴라 전극을 배치합니다. 심실의 정점 근처의 땅 ECG 전극을 배치합니다. </li></ol></li><li> 더럽히는 것 <ol><li> 형광 염료 및 전기 기계 uncoupler의 blebbistatin 모두 빛에 민감한 때문에, 어두운 방에서 아래에 설명 된 모든 절차를 수행합니다. 먼저 디메틸 설폭 시드 (2.5 mM)을 분액으로의 스톡 용액, 1.25 ㎎ / ㎖로 (30 μL를 각각) 전압 감응 염료 RH-237 또는 디 -4- ANNEPS을 준비하고 -20 ℃에서 분취 량을 저장한다. </li><li> 가온 (37 ° C) 타이로드 용액 1 ml의 염료 원액 5-10 μL 희석하고 관상 동맥 관류 라인에 주입인라인 루어 주입 포트를 사용하여 5-7 분에 걸쳐. </li><li> 사전에 원액 (디메틸 설폭 사이드 2 ㎎ / ㎖, 6.8 밀리미터)로 blebbistatin 준비하고 4 ℃에서 보관합니다. </li><li> 안정화의 20 분 후, 관류 액의 가온 0.5 ㎖ (37 ° C)를 추가하고 blebbistatin 가온 (37 ° C) 타이로드 용액 1 ㎖에 blebbistatin 0.1 mL로 희석. 이어서 인라인 루어 주입 포트를 사용하여 5-7 분에 걸쳐 관상 동맥 관류 라인에 주입. </li></ol></li></ol> 격리 된 심방 준비에서 SAN 3. 광학 매핑 <ol><li> 수단 <ol><li> 격리 된 심방 준비, 분리 및 전체 심장 준비 단계 1.1-1.5에서 상술 한 바와 같이 마음을 cannulate. </li><li> 멀리 전방 측면에서 심실을 해부하다. 그림 1A, 오른쪽 패널을 참조, 자세한 내용은 # 1를 잘라. </li><li> 삼첨판 버지니아 통해 절단하여 RA를 엽니 다TV를-SVC 축을 따라 LVE (TV). 그림 1A, 오른쪽 패널을 참조, 자세한 내용은 # 2를 잘라. </li><li> RAA를 엽니 다 crista의 terminalis의 중간 다리를 잘라. 그림 1A, 오른쪽 패널을 참조하십시오, # 3를 잘라. </li><li> 상기 RAA의 오른쪽 하단의 가장자리 위 컷 # 3의 중간 선에서 절단을 수행하여 전방 심방 자유 벽을 열고 평평 실리콘 피복 실의 바닥 심방 자유 벽에 핀. # 4 잘라도 1a에 중공의 화살표 방향 참조. 심방의 손상을 방지하기 위해 제조 피닝 심실 조직의 림 보존. </li><li> 마찬가지로, LA의 부속물 (LAA)의 MV-상단 모서리를 따라 MV 통해 절단에 의해 열린 LA. </li><li> LAA의 중간 림 근처까지 전방 심방 자유 벽을 통해 열린 LAA의 중간에서 잘라 LAA를, 엽니 다. </li><li> 전방 심방 자유 벽 ALO를 엽니 다동일한 방향 겨 후 평탄화 및 실 가드 코팅 챔버의 바닥에 고정. </li><li> 부분적으로 심방 중격 조직을 제거합니다. 이 초점에없는 조직에서 광 신호의 산란을 줄일 수 있습니다. 따라서, LA 및 RA뿐만 아니라 SAN 및 아트 리오 - 심실 접합 (AVJ)는 모두이 준비 (그림 1B)에 액세스 할 수 있습니다. </li><li> 외막과 내막 양면에서 superfusion을 허용하기 위해, 실리콘 코팅 챔버의 바닥으로부터 약 0.5 mm로 최종 제제 올려. </li><li> SVC를 근처에 집중 유량 12 ㎖ / 분 ~ 일정한 속도로 가온 함께 제제 (37 ° C) 타이로드 용액 및 Superfuse ~ 목욕 superfusion 30 ㎖ / 분. </li><li> 해부 RAA의 가장자리에 맞춤 간격의 Ag / AgCl을 2 전극 (0.25 mm 직경)을 놓습니다. 그런 다음 각각 두 개의 ECG 12mm 바늘 (29G)를 RAA 및 LAA 근처 전극 (극성)을 배치합니다. 지상 ECG 차기를 배치AVJ 근처에 탔다. </li></ol></li><li> 더럽히는 것 <ol><li> 전압에 민감한 염료 (RH-237 또는 디 - 4 ANNEPS)의 직접적인 응용 프로그램을 수행합니다. 이를 위해, 가온 (37 ° C) 타이로드 용액 1mm에서 염료 원액 1-2 μL 희석 천천히 1mm 피펫을 사용하여 제제의 표면에 희석 된 염료를 방출. </li><li> 또는, 전체 심장 준비 (단계 2.2.1-2.2.2)에 대한 전술 한 것과 유사한 랑겐 돌프 - 관류 심장의 관상 동맥 재관류를 통해 전압에 민감한 염료와 심방 염색을 수행합니다. <ol><li> 한 바와 같이 관상 동맥 관류 염색 후에 심방 준비 격리. 양호한 형광 레벨에 도달하는 데 필요한 경우 추가적인면 염색을 수행한다. 빠른 심방 분리 염료 표백하지 않고 염색 품질에 영향을 미치지 않는다. </li></ol></li><li> 전기 기계 uncoupler, blebbistatin으로 제조 고정시킨다. 상상력 3-5 μl를 희석가온 (37 ° C) 타이로드 용액 원액 bbistatin 천천히하면 준비의 표면 및 주변 솔루션을 희석 blebbistatin를 놓습니다. blebbistatin는 준비와 염색 동안 빛에 빛에 민감, 13, 14 피할 긴 노출로 표시되어 있기 때문에. </li><li> 수축을 억제하는 데 필요한만큼 추가 blebbistatin 응용 프로그램을 수행합니다. 일반적으로 3 추가 애플리케이션 (각 어플리케이션마다 3-5 μL)까지 정확하게 SAN 심방 활성화를 재구성하기 위해 충분히 동 잡음을 억제 할 수있다. </li><li> 관류에 따뜻하게 blebbistatin의 추가 0.5 ML을 추가합니다. 이 절차를 수행하는 동안, 염료 / blebbistatin는 심방 조직을 염색 할 수 있도록 30 초 동안 superfusion을 일시 중지합니다. </li></ol></li></ol> 4. 광학 매핑 설정 주 :. 광학적 맵핑 시스템의 상세한 설명은 다른 곳에서 제공된다 (12) <ol><li> 투과 전자 현미경과 카메라를 사용하여2000 프레임 / 초 이상, 집광 렌즈의 시리즈의 poral 해상도는 100 μm의 / 화소 이상의 공간 해상도를 달성한다. 이는 SAN 활성화 및 intranodal 전파를 재구성 할 필요가있다. </li><li> 진동 용액으로부터 동 잡음을 감소시키기 위해, 심장 / 절연 심방 제조 위에 상기 용액의 표면에있는 작은 커버 글라스를 고정한다. </li><li> 정전류, 저잡음 할로겐 램프에 의해 제공되는 여기 광 (520 ± 45 nm 파장)를 사용합니다. 가요 갈래 광 가이드를 사용하여 제조 상 여과 광속을 지시. </li><li> 긴 패스 필터 (&gt; 715 ㎚)에 의해 방출 된 형광 필터. 수집, 디지털화 및 카메라 제조업체에서 제공하는 컴퓨터 사용하여 소프트웨어를 획득 한 형광 신호를 저장합니다. </li></ol> 5. 데이터 처리 주 : 광 맵핑 데이터 수집 및 상이한 시점에서의 형광 강도의 행렬로 저장됩니다. 화소 represe특정 시점에서, 티슈 제조의 특정 위치에서 수집 된 형광 강도의 측정치를 NTS. 배경 형광이 자동으로 막 전압 변화에 의해 생성 및 미세 전극 시스템에 의해 측정 된 활동 전위 (APS)에 해당하는 반전 형광 변화를보다 효율적으로 시각화 할 수 있도록 픽셀 당을 제거한다. 영상 분할, 공간 필터링, 시간적 필터링하고, 기준 편차 제거를 포함한 광학 영상 데이터를 처리에서 여러 단계의 상세 포커스 리뷰가 제공된다. (15) <ol><li> 수동 또는 조직의 경계를 결정하기 위해 특정 알고리즘 (예를 들어, 임계 또는 에지 검출) (15)를 이용하여 1 (포함 화소) 0 (제외 픽셀)의 바이너리 마스크를 생성하고 시퀀스의 각 프레임에 마스크를 적용한다. 이 과정은 추가 처리를 위해 관심 영역을 강조 바이너리 이미지를 만듭니다. </li><li> 선택 해제의 신호 대 잡음비를 향상시키기iCal의 데이터는, 상기 선택된 관심 영역 내에서 신호를 필터링. 이를 위해, 예를 들어, 버터 워스, 체비 셰프 유형 및 / 또는 시간 필터링 ((잡음 데이터, × 5 5 예 3 × 3, 원하는 회선 빈 또는 커널에 의해 정의 된 이웃 형광 픽셀, 즉 평균 이상) 공간을 사용 1, 체비 세프 2 형, 타원 등) (그림 2). 데이터를 해석 할 때 염두에 여과에 의한 유물의 가능성을 유지합니다. 자세한 내용은 리뷰를 참조하십시오. (15) </li><li> 필요한 경우 (원래의 신호에 하이 패스 필터링 또는 다항식 피팅을 사용하여) 광 기록에서 기준선의 드리프트를 제거하고 0 (최소 형광) (1) (형광 극대)로부터 화소 신호를 정규화. </li><li> 단일 AP의 전파에 대한 모든 픽셀의 활성화 시간을 포함 시간 윈도우를 선택한다. F는 fluorescen입니다 최대 상향 행정 유도체의 시간 (DF / DT 최대, 각 픽셀에게 그것의 활성화 시간을 할당가전 ​​강도). 모든 화소의 활성화 시간을 사용하여, 활성화 등시 맵을 재구성. 각 아이소 크론 따라서 동시에 활성화 픽셀을 표시합니다. </li><li> 각 픽셀 (재분극, APD (80)의 80 %, 예를 들면) 재분극 특정 레벨에서 동작 시간과 시간 사이의 기간을 계산 들면 AP 기간 (APD) 분산 맵을 재구성한다. 모든 화소 APD 값을 사용하여, APD 분포 등시 맵을 재구성. 각 아이소 크론 따라서 같은 APD와 픽셀을 표시합니다. </li><li> AP의 전파에 대한 전도 속도를 계산하는 5.4에서 계산 된 활성화 시간 데이터에 제제의 표면에 장착한다. 이를 위해, 다항식 피팅 표면 또는 로컬 커널 표면 평활화를 사용하고 장착 표면의 기울기로부터 로컬 전도 속도 벡터를 구한다. </li><li> 재분극 맵을 만들려면 각 픽셀에게 그것의 재분극 시간을 할당, 최대 초 미분으로 정의 (D 2 F / DT2) OAP 또는 90 % 재분극시의 끝에서 광 신호. </li></ol>

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E., 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=Impaired+sinoatrial+node+function+and+increased+susceptibility+to+atrial+fibrillation+in+mice+lacking+natriuretic+peptide+receptor+C.">Impaired sinoatrial node function and increased susceptibility to atrial fibrillation in mice lacking natriuretic peptide receptor C.</a> J Physiol. 593 (5), 1127-1146 (2015).</li><li>Torrente, A. G., 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=Burst+pacemaker+activity+of+the+sinoatrial+node+in+sodium-calcium+exchanger+knockout+mice.">Burst pacemaker activity of the sinoatrial node in sodium-calcium exchanger knockout mice.</a> Proc Natl Acad Sci U S A. 112 (31), 9769-9774 (2015).</li><li>Lang, D., Sulkin, M., Lou, Q., Efimov, I. R. <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+action+potentials+and+calcium+transients+in+the+mouse+heart.">Optical mapping of action potentials and calcium transients in the mouse heart.</a> J Vis Exp. (55), e3275 (2011).</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. 4 (5), 619-626 (2007).</li><li>Swift, L. M., 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=Properties+of+blebbistatin+for+cardiac+optical+mapping+and+other+imaging+applications.">Properties of blebbistatin for cardiac optical mapping and other imaging applications.</a> Pflugers Arch. 464 (5), 503-512 (2012).</li><li>Laughner, J. I., Ng, F. S., Sulkin, M. S., Arthur, R. M., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+and+analysis+of+cardiac+optical+mapping+data+obtained+with+potentiometric+dyes.">Processing and analysis of cardiac optical mapping data obtained with potentiometric dyes.</a> Am J Physiol Heart Circ Physiol. 303 (7), H753-H765 (2012).</li><li>Liu, J., Dobrzynski, H., Yanni, J., Boyett, M. R., Lei, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organisation+of+the+mouse+sinoatrial+node:+structure+and+expression+of+HCN+channels.">Organisation of the mouse sinoatrial node: structure and expression of HCN channels.</a> Cardiovasc Res. 73 (4), 729-738 (2007).</li><li>Verheijck, E. E., 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=Electrophysiological+features+of+the+mouse+sinoatrial+node+in+relation+to+connexin+distribution.">Electrophysiological features of the mouse sinoatrial node in relation to connexin distribution.</a> Cardiovasc Res. 52 (1), 40-50 (2001).</li><li>Krishnaswamy, P. S., 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=Altered+parasympathetic+nervous+system+regulation+of+the+sinoatrial+node+in+Akita+diabetic+mice.">Altered parasympathetic nervous system regulation of the sinoatrial node in Akita diabetic mice.</a> J Mol Cell Cardiol. 82, 125-135 (2015).</li><li>Nygren, A., Lomax, A. E., Giles, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+action+potential+durations+in+isolated+mouse+left+and+right+atria+recorded+using+voltage-sensitive+dye+mapping.">Heterogeneity of action potential durations in isolated mouse left and right atria recorded using voltage-sensitive dye mapping.</a> Am J Physiol Heart Circ Physiol. 287 (6), H2634-H2643 (2004).</li><li>Efimov, I. R., Fedorov, V. V., Joung, B., Lin, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+cardiac+pacemaker+circuits:+methodological+puzzles+of+the+sinoatrial+node+optical+mapping.">Mapping cardiac pacemaker circuits: methodological puzzles of the sinoatrial node optical mapping.</a> Circ Res. 106 (2), 255-271 (2010).</li><li>Aschar-Sobbi, R., 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=Increased+atrial+arrhythmia+susceptibility+induced+by+intense+endurance+exercise+in+mice+requires+TNFalpha.">Increased atrial arrhythmia susceptibility induced by intense endurance exercise in mice requires TNFalpha.</a> Nat Commun. 6, 6018 (2015).</li><li>Hansen, B. 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=Atrial+fibrillation+driven+by+micro-anatomic+intramural+re-entry+revealed+by+simultaneous+sub-epicardial+and+sub-endocardial+optical+mapping+in+explanted+human+hearts.">Atrial fibrillation driven by micro-anatomic intramural re-entry revealed by simultaneous sub-epicardial and sub-endocardial optical mapping in explanted human hearts.</a> Eur Heart J. 36 (35), 2390-2401 (2015).</li><li>Lang, D., Petrov, V., Lou, Q., Osipov, G., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+control+of+heart+rate+in+a+rabbit+heart.">Spatiotemporal control of heart rate in a rabbit heart.</a> J Electrocardiol. 44 (6), 626-634 (2011).</li><li>Kanlop, N., Sakai, T. <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+study+of+blebbistatin-induced+chaotic+electrical+activities+in+isolated+rat+atrium+preparations.">Optical mapping study of blebbistatin-induced chaotic electrical activities in isolated rat atrium preparations.</a> J Physiol Sci. 60 (2), 109-117 (2010).</li><li>Glukhov, A. V., Flagg, T. P., Fedorov, V. V., Efimov, I. R., Nichols, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+K(ATP)+channel+pharmacology+in+intact+mouse+heart.">Differential K(ATP) channel pharmacology in intact mouse heart.</a> J Mol Cell Cardiol. 48 (1), 152-160 (2010).</li><li>Wu, 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=Altered+sinoatrial+node+function+and+intra-atrial+conduction+in+murine+gain-of-function+Scn5a+/DeltaKPQ+hearts+suggest+an+overlap+syndrome.">Altered sinoatrial node function and intra-atrial conduction in murine gain-of-function Scn5a+/DeltaKPQ hearts suggest an overlap syndrome.</a> Am J Physiol Heart Circ Physiol. 302 (7), H1510-H1523 (2012).</li></ol>

No conflicts of interest are declared.

랑겐 돌프 - 관류 온 마음 1) 그대로 SAN, 2) SAN은 고립에, 심방 준비를 개설 : 여기, 우리는 마우스 SAN 준비의 두 가지 유형을 제시했다. 준비의이 두 종류의 서로 다른 실험 목적을 제공합니다. 랑겐 돌프 - 관류 온 마음 준비에서 그대로 심방 구조는 가능한 재진입 빈맥 성 부정맥 동안 SAN과 심방 사이의 복잡한 심방의 이러한 심방 세동과 같은 부정맥뿐만 아니라 상호 작용을 연구 할 수있는 보존됩?...

Novel scientific breakthroughs that lead to leaps in the understanding of human physiology are often preceded by technological advances. Fluorescent optical mapping, for example, enables investigation of multiple physiological parameters in both cells and tissues.1,2 It significantly improved our understanding of how the anatomical structure is associated with electrophysiological functions and dysfunctions. In the heart, the natural pacemaker and conduction systems such as the sinoatrial node (SAN) and the atrioventricular junction consist of nodal myocytes that are insulated by the surrounding atrial myocytes.3-5 Such organization creates complex three-dimensional structures with specialized electrical properties. Both structural and functional remodeling of these pacemaker structures has been recognized to form significant electrophysiological heterogeneities.6,7 Understanding the mechanism underlying how such heterogeneities result in SAN dysfunctions and atrial arrhythmogenesis will dramatically benefit the clinical treatment of these diseases. It requires a technique to visualize the propagation of electrical signals at the tissue level, such as optical mapping. 
Recently, accumulating evidence has proven the advance of optical mapping in studies of atrial electrophysiology and pathology.2 However, novel and rigorous studies are dependent on the accurate interpretation of experimental data, whose validity and stability rely on careful experiment protocols. Genetically modified mice are extensively used for research as animal models of human diseases including sick sinus syndrome, pacemaker abnormalities and atrial arrhythmias.6,8-11 Thus, a combination of fluorescent optical mapping with transgenic mouse models provides a powerful tool to study cardiac electrical abnormalities associated with various pathologies. In this paper, we present a protocol for high-resolution optical mapping of the mouse SAN and atrium. Specifically, we discuss and compare different dye loading approaches, time effects on dye bleaching and heart rate stability during the experiment.

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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.

랑겐 돌프 - 관류 심장에서 그대로 SAN의 광학 매핑 자연 동 율동에 대한 재구성 RA 활성화 등고선의 전형적인 예는 랑겐 돌프 - 관류 마우스 심장 그림 3에 표시됩니다. 초기 활성 점은 SAN 해부학 정의 SVC 근처 intercaval 영역 내에 위치한다. 1.0 및 0.5 msec의 샘플링 레이트에서 취득 3,16 개의 RA 활성화 등고선지도는도 3b에 도?...

Watch this Scientific Journal Video about High-resolution Optical Mapping of the Mouse Sino-atrial Node at JoVE.com

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

여기에, 우리는 높은 공간 및 시간 해상도, 마우스 우심방 특히 중국 - 심방 노드에서 전기 활동의 광학 매핑을위한 프로토콜을 제시한다.

마우스 중국 - 심방 노드의 고해상도 광학 매핑

Sino-atrial node (SAN) dysfunctions and associated complications constitute important causes of morbidity in patients with cardiac diseases. The ...

high-resolution-optical-mapping-of-the-mouse-sino-atrial-node

Research

JoVE Journal

Biology

15.5K 조회수.  University of Wisconsin-Madison School of Medicine and Public Health.  여기에, 우리는 높은 공간 및 시간 해상도, 마우스 우심방 특히 중국 - 심방 노드에서 전기 활동의 광학 매핑을위한 프로토콜을 제시한다. 

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

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

Drosophila의 유충에서 냄새 기반 행동의 고해상도 측정

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

연구

생물학

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 ...

Major Components of the Light Microscope

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

라이트 현미경의 주요 구성 요소

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for ...

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

성인 Drosophila Melanogaster의 로코의 고해상도 비디오 추적

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

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 ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

유도 만능의 줄기 세포에서 파생 마우스의 생성

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

공장 Vasculature의 3 차원 구조 및 기능을 시각화하기 위해 높은 해상도로 계산 된 단층 촬영을 사용하여

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

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 ...

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.

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 ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

고해상도 시간 경과 이미징 및 생활 인간 제대 정맥 내피 세포의 미세 소관 역학의 자동 분석

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...