이 연구는 교육 과학 기술부 (2013068067)에 의해 투자 한국 연구 재단 (NRF)를 통해 기초 과학 연구 프로그램에 의해 지원되었다; 교육 과학 기술부, 서울 대학교 병원, 고혈압 학회 (2013), SK 텔레콤 연구 기금의 한국 교육부의 두뇌 한국 (21) 대학원 프로그램에 의해 (NO. 3420130290)를 중국의 국립 자연 과학 재단 (NSFC 31460265; NSFC 81260035).

프로토콜은 건강의 UN 국립 연구소에 의해 게시 된 실험 동물의 관리 및 사용에 대한 가이드에 따라입니다 (NIH 공개 번호 85-23, 1996 개정). 그것은 서울 대학교의 기관 동물 관리 및 사용위원회 (IACUC) (IACUC 승인 번호 : SNU-101213-1)에 의해 승인되었다. 1. 버퍼 준비 (표 재료 및 장비) <ol><li> mm 단위 (실험 당일 신선한 분리 용액 300㎖를 준비 : 염화나트륨, 135의 KCl, 5.4;의 MgCl 2, 3.5, 글루코스, 5; HEPES, 5; 나 2 HPO 4, 0.4, 타우린 20] pH가 7.4 NaOH로). </li><li> 밀리미터에 (실험의 날에 신선한 스토리지 솔루션 100 ml의 준비 : 염화나트륨 (120);의 KCl 5.4, 황산 5, CaCl2를 0.2, 나트륨 피루 베이트 (5); 5.5 포도당, 타우린 (20), HEPES (10), 만니톨 (29), pH 7.4의 ) NaOH로. </li><li> 밀리미터의 관류 액 1L (준비 : 염화나트륨, 141.4을, KCl을, 4;의 NaH 2 PO 4, 0.33; MGC를L이 1; HEPES, 10; 포도당, 5.5; 염화칼슘 2, 1.8; 만니톨, 14.5; pH를 NaOH로 7.4). </li><li> 절연 25 ㎖로 콜라게나 제 (1 ㎎ / ㎖), 프로테아제 (0.1 ㎎ / ㎖), 소 혈청 알부민 (BSA, 1.67mg / ㎖) 및 칼슘 (0.05 mm)를 첨가하여 콜라겐 용액 1 30 ㎖를 준비 해결책. </li><li> 차단 용액을 16.7 ml의 (1 ㎎ / ㎖) BSA (1.67 ㎎ / ㎖), 및 칼슘 (0.05 ㎜) 콜라게나 제를 첨가하여 콜라겐 용액이 20 ㎖를 준비한다. </li><li> 차단 용액을 40 ml의 BSA 0.4 g을 첨가하여 BSA 용액 40 ml를 준비. 10 mL 및 두 개의 비커에 30 ml의 분리. 칼슘을 BSA 용액 2+ 30 mL로 추가되도록 BSA 용액의 최종 칼슘 농도가 1 mm이다. </li></ol> 좌심실 (LV) 심근 세포의 분리 2. 준비 <ol><li> 준비와 격리 방에 동물 시설에서 깨끗한 전송 케이지 8-12 주령, 수컷 스프 라그 - 돌리 (SD) 쥐를 전송합니다. </li><li> H37 오 C. 두 개의 수조를 먹고 참고 : 물에 대해 하나의 수조를 사용 - 재킷 저수지와 랑겐 돌프 관류 시스템의 관류 관. 하나의 근육 세포를 분리하기 위해 심근 조직 트렁크를 선동하기 위해 다른 수조를 사용합니다. </li><li> 각각 30 mL 및 20 ㎖에 볼륨을 만들기 위해 5 ㎖의 콜라겐 액 (1) 및 16.7 ml의 콜라겐 액이 25 ml의에 (칼슘없이 2+) BSA 용액의 3.3 ML을 추가합니다. </li><li> 랑겐 도르프 재관류에있어서, 각각 (도 1a)에서 분리 용액 (100 mL) 및 1 열 (골 1) 및 열 (2)에 콜라게나 제 용액 1 (30 ㎖) (골 2)를 추가한다. </li><li> 랑겐 돌프 관류 시스템 (도 1A)의 산소 연결 튜브를 통해 골 1 Col.2의 분리 용액과 콜라겐 용액 1 네이트. 마찬가지로 네이트 콜라게나 제 용액과 두 비아 100 % O 2를 진탕 수욕에서 BSA 용액산소 접속 관. </li></ol> LV 근육 세포 3. 분리 <ol><li> 펜토 바르 비탈 나트륨 (30 ㎎ / ㎏)의 복강 내 주사를 통해 SD 쥐를 마취하고 곤란 발가락과 철수 반사의 부족으로 마취 상태를 확인합니다. </li><li> 해부 트레이에 쥐를 이동합니다. 앙와위에서 테이프 본체의 측면에 4 개의 다리를 고정. </li><li> 심장이 손상되지 않도록 보장, 가슴을 열고 수술 가위 흉부 중간 축 절개를 적용합니다. 연결 용기 (예를 들어, 우수하고 하대 정맥, 폐 혈관 및 대동맥) 및 심낭 막 (13)로부터 심장을 해부 깨끗한 가위의 또 다른 한 쌍을 사용합니다. </li><li> 충분한 길이의 대동맥의 일부를 남기기 (5-8mm)를 미세 집게로 대동맥 클램프, 빠르게 1 분 (그림 1A) 내에서 랑겐 돌프 관류 시스템의 캐 뉼러를 탑재합니다. 대동맥을 통해 단단히 봉합 나사 4/0을 묶어. </li><l난&gt; 골 1 밸브를 켜고 10 분 동안 예열 및 산소 격리 솔루션 (관류 속도 : 12 ~ 14 ml / 분)와 고립 된 마음을 관류. , 골 ​​1 밸브의 전원을 끄고 골 2의 밸브를 켜고 8 콜라겐 액 (1)와 함께 마음을 관류 - 10 분. </li><li> 대동맥을 절단하여 소화 마음을 분리하고 신선한 격리 솔루션 (그림 1Bi)를 포함하는 플라스크에 집게로 대동맥을 유지하여 전송할 수 있습니다. 작은 조각 (~ 22mm, 그림 1Bii)에 (격막 포함) LV 무료 벽의 대부분을 잘라 미세 가위와 집게를 사용합니다. </li><li> 미리 예열 및 산소 신선한 콜라게나 제 용액 (2) (그림 1Biii)를 포함하는 플라스크에 조각을 전송합니다. 10 분 동안 흔들어. 심근 함유 콜라겐 용액 2에 산소를 전달하는 것. </li><li> (BSA 용액을 함유 - 2+ 흡입 전구 적기를 사용하여 10 mL의 원심 분리 튜브에 현탁 및 CA 추가 - 심근 이동1 : 볼륨 1). 2 분 30 g에서 원심 분리하고 상층 액을 버린다. BSA 용액 5 ㎖에 심근 세포 펠렛을 다시 일시. 원심 분리기 뜨는을 버리고. </li><li> 심근 세포 펠렛을 분산 및 RT (그림 1Biv-VI)에서 미리 산소 저장 용액 10 ml의 근육 세포를 유지. </li><li> 플라스크에 남아있는 LV 조직으로 - 절차를 (3.9 3.7 단계) 반복합니다. 좌심실 조직의 대부분이 근육 세포의 좋은 수율을 얻을 사라질 때까지 다시 - (3.9 3.7 단계)의 절차를 반복하십시오 :합니다. </li><li> 실험이 끝날 때까지 실온에서 6-8 시간 동안 스토리지 솔루션의 근육 세포를 유지합니다. </li></ol> 세포 내 칼슘 과도과 심근 수축 4. 동시 측정 <ol><li> 아세 톡시 메틸 에스테르의 Fura-오전 2시 (2 μM)와로드 LV의 근육 세포. 참고 : 어두운 튜브 (그림 1Bvii)에로드 된 근육 세포로 모든 로딩 절차와 실험을 수행합니다. <ol><li> 하기 Centr10 초 동안 2,000 XG에서 심근 서스펜션 (1 ml)에 ifuge. 뜨는을 취소하고 낮은 칼슘 농도 (250 μM, 표 재료 및 장비)와 타이로드 용액 1 ㎖의 심근 세포 펠렛을 다시 일시 중지합니다. </li><li> 부드럽게 심근 세포 현탁액을 분산,은 Fura - 오전 2시 및 폴록 사머 407 (2 μL)를 추가하고, 실온에서 혼합물을 유지 - 15 분 (20 ~ 24 O를 C) (그림 1Bvii). </li><li> 원심 혼합물을 5 초 동안, 상층 액을 제거하고 500 μM 칼슘을 함유하는 관류 액 1ml에 심근 세포 펠렛을 분산. 10 분 후, 5 초 동안 혼합물을 원심 분리하고 상층 액을 버린다. </li><li> 신선한 관류 용액 (500 μM 칼슘, 1 ml)에 추가하고의 Fura - 오전 2시 유지 - 녹음이 솔루션에로드 된 심근 세포 펠렛을. </li></ol></li><li> 좌심실 심근의 수축과 세포 내 칼슘의 측정 2 + <ol><li> 기록하기 전에 통해 실행 관류 관을 채우기인 타이로드 용액, 물 재킷 (36) 오 C.에 미리은 예열 </li><li> 8 분 - 5 거꾸로 형광 현미경의 챔버에로드 좌심실 심근 서스펜션 - 상기의 Fura 오전 2시의 몇 방울을 배치합니다. 천천히 타이로드 용액 (2.2 ㎖ / 분)를 관류. </li><li> 필드 자극 (2 Hz에서) 시작하는 디지털 자극의 전면 패널에있는 &quot;시작&quot;버튼을 누릅니다. 주 : 출력 전압 (10 V는 5 밀리 초 지속 기간)는 상기 챔버의 양측에 위치하는 백금 와이어를 통해 챔버에서 심근 세포에 적용된다. <ol><li> 안정적으로 그 계약을 선택 심근 세포 기록 (하이퍼 또는 저자 극성 수축을 표시하지 않는 것). </li></ol></li><li> 비디오 기반 근절 검출 시스템의 수평 위치에서의 선택의 근세포를 조정 근섬유 분절 최적의 이미지를 획득하기 위해 상기 현미경의 초점을 조절한다. 분명 근섬유 분절는 누가 뭐라고해도 평균 때까지 관찰되는 영역에 보라색 사각형 상자를 배치근절 길이 (적색 피크)의 단수 날카로운 피크 (그림 2A, 낮은 이미지)를 표시합니다. </li><li> 필드 자극 (도 2A와 B)에 응답하여 근절 길이의 변화를 기록한다. 주 : 1 내에서로드 된 근육 세포를 사용 - 2 시간. </li><li> 비디오 기반 근절 검출 시스템의 필드가 근세포 (도 2A)의 크기가되도록 카메라의 조리개를 조정한다. 510 nm의 (인수 주파수 1000 Hz에서)에서 360 나노 미터 / 380 nm에서 여기 및 방출과 심근을 자극한다. 기록 근절 단축 및 현장 자극 (그림 2B)로 Fura2 AM 비율. </li></ol></li></ol> Myofilament 칼슘 감도 5. 평가 <ol><li> 측정 값이 모두 (- (20 추적 10)과 같은 심근의 근절 길이 대의 Fura-2 비율의 위상면 루프를 플롯 근절 길이 및 정상 상태에서 2 + 과도 칼슘 평균s와 델타의 변화도 2C). </li><li> 각각의 플롯에서 50 % 완화에서의 Fura -2 비율 (EC 50, 그림 2C)을 정의합니다. 루프 각 개입의 EC (50) 양을 비교합니다. </li></ol>

<ol>
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C., 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=Functional+effects+of+the+DCM+mutant+Gly159Asp+troponin+C+in+skinned+muscle+fibres.">Functional effects of the DCM mutant Gly159Asp troponin C in skinned muscle fibres.</a> Pflugers Arch. 453 (6), 771-776 (2007).</li><li>Willott, R. H., 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=Mutations+in+Troponin+that+cause+HCM,+DCM+AND+RCM:+what+can+we+learn+about+thin+filament+function?.">Mutations in Troponin that cause HCM, DCM AND RCM: what can we learn about thin filament function?.</a> J Mol Cell Cardiol. 48 (5), 882-892 (2010).</li><li>Yasuda, S. I., 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=A+novel+method+to+study+contraction+characteristics+of+a+single+cardiac+myocyte+using+carbon+fibers.">A novel method to study contraction characteristics of a single cardiac myocyte using carbon fibers.</a> Am J Physiol Heart Circ Physiol. 281 (3), H1442-H1446 (2001).</li><li>Sears, C. 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=Cardiac+neuronal+nitric+oxide+synthase+isoform+regulates+myocardial+contraction+and+calcium+handling.">Cardiac neuronal nitric oxide synthase isoform regulates myocardial contraction and calcium handling.</a> Circ Res. 92 (5), e52-e59 (2003).</li><li>Ashley, E. A., 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=Cardiac+nitric+oxide+synthase+1+regulates+basal+and+beta-adrenergic+contractility+in+murine+ventricular+myocytes.">Cardiac nitric oxide synthase 1 regulates basal and beta-adrenergic contractility in murine ventricular myocytes.</a> Circulation. 105 (25), 3011-3016 (2002).</li><li>Zhang, Y. H., 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=Reduced+phospholamban+phosphorylation+is+associated+with+impaired+relaxation+in+left+ventricular+myocytes+from+neuronal+NO+synthase-deficient+mice.">Reduced phospholamban phosphorylation is associated with impaired relaxation in left ventricular myocytes from neuronal NO synthase-deficient mice.</a> Circ Res. 102 (2), 242-249 (2008).</li><li>Roe, M. W., Lemasters, J. J., Herman, B., 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=Assessment+of+Fura-2+for+measurements+of+cytosolic+free+calcium.">Assessment of Fura-2 for measurements of cytosolic free calcium.</a> Cell Calcium. 11 (2-3), 63-73 (1990).</li><li>Grynkiewicz, 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=A+new+generation+of+Ca2++indicators+with+greatly+improved+fluorescence+properties.">A new generation of Ca2+ indicators with greatly improved fluorescence properties.</a> J Biol Chem. 260 (6), 3440-3450 (1985).</li></ol>

The authors declare that they have no competing financial interests.

여기서 우리는 하나의 고립 된 심장 근세포에 myofilament 칼슘 감도의 변화를 평가하기 위해 프로토콜을 설명하고이 전기 생리 특성과 함께 매개 변수, 세포 내 칼슘 2 + 과도 및 myofilament 역학 측정의 중요성을 강조한다. 하나 또는 두 개의 매개 변수의 녹음은 심장의 수축과 이완을 기본 메커니즘을 해명하지 않을 수 있기 때문입니다. 심근의 수축과 세포 내 칼슘 2 + 프로필 ...

여기 심장 수축 (EC) 결합 심근 즉, 심장의 수축 1,2- 함수의 기계적 특성을 분석하는 기본 방식이다. EC 커플 sarcolemmal이 이온 채널의 활성에 보조 막을 탈분극에 의해 개시된다 (예를 들어, 패치 클램프 기술을 통해 측정 할 수있는 전압 - 게이트 나 + 채널). (나노 몰에서 nm의 세포질 칼슘 농도를 증가 ryanodine 수용체 (RyRs)를 통해 전압 게이트 L 형의 후속 활성화 칼슘은 2 + 채널 LTCCs를 통해 2+ 유입이 칼슘의 대부분을 트리거 (LTCCs)과 칼슘 2 + 릴리스, )는 (μM) 수준을 마이크로합니다. 세포질 칼슘의 이러한 증가는 얇은 필라멘트에 트로포 닌 C (TNC)에 결합하는 2 + 칼슘을 촉진하고 이끌어 필라멘트 복합체의 구조적 변화를 액틴 - 미오신 상호 작용을 촉진하고 myocardi을 달성알은 3 수축. 반대로, 세포질 칼슘은 SR에서 칼슘 2 + -ATPase를 통해 근질 세망 (SR) (SERCA2a)로 다시 재 uptaken거나 나 + / 칼슘 교환기 및 plasmalemmal를 통해 심근에서 압출 칼슘 2 + ATPase의 1, 2. 따라서, 세포질 칼슘의 감소는 2 + 액틴 - 미오신과 심근 이완 1-3의 분리의 결과로, 다시 원래 상태로 얇은 필라멘트의 구조적인 변화를 선동. 대부분의 포유 동물의 심장 세포 1 세포질 칼슘 제거의 90 % - 이러한 방식에서, SERCA2a의 활성은 일반적으로 70를 차지하기 때문에 심근 완화의 속도를 결정하는데 고려된다. 등 LTCC, RyR 및 SERCA2a에 의해 같은 비정상적인 칼슘 처리로 병에 걸린 심장 1-4에서 장애인 수축과 이완의 기본 메커니즘을 고려하고있다. 2 + 총 세포 내 칼슘의 약 1 %에 대한 EC 커플 링 계정의 메신저와 칼슘의 대부분 같은 기능은 세포 내 칼슘 2 + 버퍼 5,6에 바인딩 무료 세포질 칼슘. 이는 다양한 칼슘 버퍼의 SR에 예를 들어, 막 인지질, ATP, 크레아틴 인산, 칼 모듈 린, parvalbumin, 근원 섬유 TNC, 미오신, SERCA2a 및 calsequestrin 심장 근육 세포에 풍부 사실이다. 5,6,7. 그 중, SERCA2a 및 TNC가 지배적 인 칼슘 2 + 버퍼 5,6,7입니다. 또한, 칼슘 2 +의 버퍼 바인딩 트 동안 역동적 인 과정이며, 추가 원인을 결합 칼슘의 변화 2+ (예를 들어, 2 + 칼슘의 30 % ~ 50 %는 2 + 과도 7 TNC에 결합 칼슘 동안 그것에서 해리) 진한 2+ 세포 내 칼슘의 변화에, 세포질 무료 칼슘의 결과를 &quot;해제&quot;entration. 그 결과, 세포 내 칼슘 수준의 교란은 수축 기능 장애 및 부정맥 8,9의 전구체입니다 이상 myofilament의 움직임을 유도한다. (생리 및 병리 모두) 많은 요인 myofilament 칼슘 버퍼링 및 myofilament 칼슘 감도 8-10 영향을 myofilament 단백질의 전사 후 변형의 원천이 될 수 있습니다. 최근에는 칼슘 2 + 과도, 비정상적인 칼슘 2 + 릴리스 및 부정맥 8의 일시 정지에 의존 증강을 유발, myofilament 단백질의 돌연변이가 칼슘 결합 친화 및 세포 내 칼슘 처리를 늘리는 것이보고되었다. 이 개념에 맞춰, 우리는 또한 높은 이완기 및 수축기 칼슘 수준과 연관된 신경 세포의 산화 질소 합성 효소의 업 규제 이차 고혈압 쥐의 마음이 myofilament 칼슘 탈감작을 보여 주었다결과적으로, 칼슘 의존적 불 활성화 (12)에 LTCC의 취약성을 증가 11. 따라서, myofilament 칼슘 감도는 세포 내 칼슘 항상성 및 심근 수축 기능의 &quot;활성&quot;레귤레이터이다. 그것은 myofilament와 칼슘 사이의 상호 작용을 분석 할 필요가있다 2+ 심근 EC 커플 링 및 심장 기능의 철저한 조사를 위해 단백질을 처리. 여기, 우리는 고립 된 심장 근육 세포에 myofilament 칼슘 감도의 변화를 평가하는 프로토콜을 설명합니다. 세포 내 칼슘 2 + 프로필의 종합 분석, myofilament 칼슘 감도 및 수축 새로운 메커니즘을 기본 심근 역학을 발굴합니다.

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

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

심부전 및 심장 부정맥은 전 세계적으로 사망률과 이환율의 주요 원인입니다. 그러나, 병에 걸린 마음의 병인과 심근 고장의 메커니즘은 완전히 명확 일이다. 최근 강력한 증거가 myofilament 칼슘 2 + 감도의 변화가 심장 근육 세포에서 세포 내 칼슘 항상성 및 이온 채널 활동에 영향을 미칠 것으로 보여, 건강하고 병에 걸린 마음에 심장 활동 전위과 수축을 담당하는 핵심 메커니즘. 사실, (예를 들어, 나 +, 칼슘 2 +와 K + 채널과 나 + -ca 2+ 기) 및 세포 내 칼슘 2 + 처리 단백질 (예., ryanodine 수용체와 칼슘을 심장 활동 전위를 기본 이온 채널의 활동 및 운송 근질 세망 (SERCA2a) 또는 phospholamban과 인산화)에서 2 + -ATPase 종래 evalu 측정된다심장 자극 - 수축 (EC) 커플 링의 기본 메커니즘을 먹었다. myofilament이 수축과 이완의 기능 부이고, myofilament 칼슘 감도 근원 섬유 성능의 구현에 필수적인 반면, 멤브레인 및 세포 내 칼슘의 변화 모두 전기 활동은 EC 결합의 트리거 신호이다. 이 연구의 초점이 아닌 그럼에도 불구하고, 몇몇 연구는 심근의 기능 분석에 myofilament 칼슘 감도를 통합합니다. 여기, 우리는 근절 단축 / 재 연장과의 Fura-오전 2시 (비율 계량 감지)와 쥐 마음에서 심장 근육 세포에 myofilament 칼슘 감도의 변화를 평가를 사용하여 세포 내 칼슘 수준을 측정하는 프로토콜을 설명합니다. 주요 목적은 칼슘 감도 역학적 분석 EC 결합에서 고려되어야한다는 myofilament을 강조하는 것이다. 난의 종합 조사번역 및 치료 가치보다 효과적인 전략을 설계에 대한 유용한 정보를 제공 할 것이다 건강하고 병에 걸린 마음에서 심근 수축력의 기초 채널, 이온 수송, 세포 내 칼슘 처리 및 myofilament 칼슘 감도에.

LV의 근육 세포는 정상 및 고혈압 쥐의 마음에서 격리됩니다. 필드 자극에 응답 분명 줄무늬 (근섬유 분절을 대표하는) 안정 수축과 막대 모양의 근육 세포가 최적의 근육 세포로 간주하고 녹음 (그림 2A) 선택됩니다. F의 igure 2a에 도시 된 예에서의 Fura 오전 2시 좌심실 심근 수평 위치와 근세포는 기록 필드 최소 배경 영역의 대부분이 포함되어 차?...

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

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

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

Myofilament 칼슘의 평가<sup&gt; 2 +</sup&gt; 감도 근본적인 심장 여진 수축 커플 링

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

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JoVE Journal

Biology

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

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

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

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

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

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

격리 된 심 실 Cardiomyocytes에 Sarcoplasmic 그물 칼슘 예비 및 세포내 확장기 칼슘 제거의 평가

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

연구

의학

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

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

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

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

동시 브라이트필드, 형광, 및 수축 심장 Trabeculae Ex Vivo의 광학 일관성 지형 이미징

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

생체공학

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

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

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

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

절연 골격근의 수축성, Fatigability 및 Alternans의 예 VIVO 평가

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

생물학

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

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

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

칼슘의 평가는 그대로 골격 근육 섬유에서 불꽃

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

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring apparatus and a motor to perform functional studies. These studies will allow measurement of cardiomyocyte stiffness (passive force) and its activation with different calcium (Ca2+)-containing solutions to determine, amongst others: maximum force development, myofilament Ca2+-sensitivity (pCa50), cooperativity (nHill) and the rate of force redevelopment (ktr). This method also enables determination of the effects of drugs acting directly on myofilaments and of the expression of exogenous recombinant proteins on both active and passive properties of cardiomyocytes. Clinically, skinned cardiomyocyte studies highlight the pathophysiology of many myocardial diseases and allow in vitro assessment of the impact of therapeutic interventions targeting the myofilaments. Altogether, this technique enables the clarification of cardiac pathophysiology by investigating correlations between in vitro and in vivo parameters in animal models and human tissue obtained during open heart or transplant surgery.

This protocol aims to describe step-by-step the technique of extraction and assessment of cardiac function using skinned cardiomyocytes. This methodology allows measurement and acutemodulation of myofilament function using small frozen biopsies that can be collected from different cardiac locations, from mice to men.

피부 심근세포를 이용한 심장 기능의 체외 평가

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring ...

Electromechanical Assessment of Optogenetically Modulated Cardiomyocyte Activity

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

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

광유전학적으로 변조된 심근세포 활동의 전기기계적 평가

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

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

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

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

마우스의 심장 수축력의 조사에 대 한 격리 유두 근육의 수축력 측정

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

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

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

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

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

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 ...

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

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

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

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

설치류 근육 세포에서 심장 수축 기능 장애 및Ca 2+ 과도 현상 분석

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

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

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

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

Myofilament 칼슘의 평가

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