Name: in vitro assessment of cardiac function using skinned cardiomyocytes
Uploaded: 2020-06-22T13:00:00.000+00:00
Duration: 8 min 19 s
Description: Watch this Scientific Journal Video about In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes at JoVE.com

저자는 과학 기술 (FCT), 유럽 연합( EU), 쿼드로 드 Referência 에스트라테기코 나시오날 (QREN), 푼도 유럽 유 데센볼비멘토 지역 (FEDER) 및 Programa Operacional Factores 드 Competitividade (경쟁) UnIC (UID / IC / 00051/ 2013) 연구 단위에 대한 포르투갈어 재단에 감사드립니다. 이 프로젝트는 경쟁 2020 - 프로비타 오페라 경쟁 E Internacionaléação (POCI), 프로젝트 DOCNET (NORTE-01-0145-FEDER-000003) 포르투갈 2020 파트너십 계약에 의해 지원 , 포르투갈 2020 파트너십을 통해 FEDER에 의해 지원됩니다 유럽 지역 개발 기금(ERDF)을 통해 리스본의 지역 운영 프로그램인 2020년 유럽 구조 및 투자 펀드가 지원하는 프로젝트 NETDIAMOND(POCI-01-0145-FEDER-016385)를 통해. Patrícia 로드리게스FCT (SFRH / BD / 96026/2013)에 의해 투자되었고 주앙 알메이다 - 코엘호는 유니버시다데 두 포르투 / FMUP 및 FSE - 펀도 사회 유럽u에 의해, NORTE 2020-Programa Operacional 지역 도 노르테, (NORTE-08-5369-FSE-000024-Programas Doutorais).

<ol>
	<li>Leite-Moreira, A. 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=Stretch-induced+compliance:+a+novel+adaptive+biological+mechanism+following+acute+cardiac+load.">Stretch-induced compliance: a novel adaptive biological mechanism following acute cardiac load.</a> Cardiovascular Research. 114 (5), 656-667 (2018).</li><li>Falcao-Pires, I., Fontes-Sousa, A. P., Lopes-Conceicao, L., Bras-Silva, C., Leite-Moreira, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+myocardial+stiffness+by+&#946;-adrenergic+stimulation+-+its+role+in+normal+and+failing+heart.">Modulation of myocardial stiffness by &#946;-adrenergic stimulation - its role in normal and failing heart.</a> Physiological Research. 60 (4), 599-609 (2011).</li><li>Cokkinos, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Introduction to Translational Cardiovascular Research. , 371-387 (2015).</li><li>van der Velden, J., Stienen, G. J. 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+Disorders+and+Pathophysiology+of+Sarcomeric+Proteins.">Cardiac Disorders and Pathophysiology of Sarcomeric Proteins.</a> Physiological Reviews. 99 (1), 381-426 (2019).</li><li>Garnier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attachment+procedures+for+mechanical+manipulation+of+isolated+cardiac+myocytes:+a+challenge.">Attachment procedures for mechanical manipulation of isolated cardiac myocytes: a challenge.</a> Cardiovascular Research. 28 (12), 1758-1764 (1994).</li><li>Brady, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+isolated+cardiac+myocytes.">Mechanical properties of isolated cardiac myocytes.</a> Physiological Reviews. 71 (2), 413-428 (1991).</li><li>Falcao-Pires, I., Leite-Moreira, A. F., Cokkinos, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+20.">Chapter 20.</a> Introduction to Translational Cardiovascular Research. 20, 371-387 (2015).</li><li>Liang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Teaching+calcium-induced+calcium+release+in+cardiomyocytes+using+a+classic+paper+by+Fabiato.">Teaching calcium-induced calcium release in cardiomyocytes using a classic paper by Fabiato.</a> Advances Physiology Education. 32 (1), 1-10 (2008).</li><li>Roche, S. M., Gumucio, J. P., Brooks, S. V., Mendias, C. L., Claflin, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+Maximum+Isometric+Force+Generated+by+Permeabilized+Skeletal+Muscle+Fibers.">Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers.</a> Journal of Visualized Experiments. (100), e52695 (2015).</li><li>Huxley, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+structure+and+theories+of+contraction.">Muscle structure and theories of contraction.</a> Progress Biophysics and Biophysical Chemistry. 7, 255-318 (1957).</li><li>Sequeira, 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=Synergistic+role+of+ADP+and+Ca(2+)+in+diastolic+myocardial+stiffness.">Synergistic role of ADP and Ca(2+) in diastolic myocardial stiffness.</a> Journal Physiology. 593 (17), 3899-3916 (2015).</li><li>Edes, I. F., 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=Rate+of+tension+redevelopment+is+not+modulated+by+sarcomere+length+in+permeabilized+human,+murine,+and+porcine+cardiomyocytes.">Rate of tension redevelopment is not modulated by sarcomere length in permeabilized human, murine, and porcine cardiomyocytes.</a> American Journal Physiology Regulatory Integrative Comparative Physiology. 293 (1), R20-R29 (2007).</li><li>King, N. 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=Mouse+intact+cardiac+myocyte+mechanics:+cross-bridge+and+titin-based+stress+in+unactivated+cells.">Mouse intact cardiac myocyte mechanics: cross-bridge and titin-based stress in unactivated cells.</a> Journal General Physiology. 137 (1), 81-91 (2011).</li><li>Hamdani, N., 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=Myocardial+titin+hypophosphorylation+importantly+contributes+to+heart+failure+with+preserved+ejection+fraction+in+a+rat+metabolic+risk+model.">Myocardial titin hypophosphorylation importantly contributes to heart failure with preserved ejection fraction in a rat metabolic risk model.</a> Circulation Heart Failure. 6 (6), 1239-1249 (2013).</li><li>Miranda-Silva, D., 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=Characterization+of+biventricular+alterations+in+myocardial+(reverse)+remodelling+in+aortic+banding-induced+chronic+pressure+overload.">Characterization of biventricular alterations in myocardial (reverse) remodelling in aortic banding-induced chronic pressure overload.</a> Scientific Reports. 9 (1), 2956 (2019).</li><li>Rodrigues, P. 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=Early+myocardial+changes+induced+by+doxorubicin+in+the+nonfailing+dilated+ventricle.">Early myocardial changes induced by doxorubicin in the nonfailing dilated ventricle.</a> American Journal Physiology Heart Circulatory Physiology. 316 (3), H459-H475 (2019).</li><li>van der Velden, 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=Alterations+in+myofilament+function+contribute+to+left+ventricular+dysfunction+in+pigs+early+after+myocardial+infarction.">Alterations in myofilament function contribute to left ventricular dysfunction in pigs early after myocardial infarction.</a> Circulation Research. 95 (11), e85-e95 (2004).</li><li>Wakili, 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=Multiple+potential+molecular+contributors+to+atrial+hypocontractility+caused+by+atrial+tachycardia+remodeling+in+dogs.">Multiple potential molecular contributors to atrial hypocontractility caused by atrial tachycardia remodeling in dogs.</a> Circulation: Arrhythmia Electrophysiology. 3 (5), 530-541 (2010).</li><li>Ait Mou, Y., le Guennec, J. Y., Mosca, E., de Tombe, P. P., Cazorla, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+contribution+of+cardiac+sarcomeric+proteins+in+the+myofibrillar+force+response+to+stretch.">Differential contribution of cardiac sarcomeric proteins in the myofibrillar force response to stretch.</a> Pflugers Archiv. 457 (1), 25-36 (2008).</li><li>Falcao-Pires, 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=Diabetes+mellitus+worsens+diastolic+left+ventricular+dysfunction+in+aortic+stenosis+through+altered+myocardial+structure+and+cardiomyocyte+stiffness.">Diabetes mellitus worsens diastolic left ventricular dysfunction in aortic stenosis through altered myocardial structure and cardiomyocyte stiffness.</a> Circulation. 124 (10), 1151-1159 (2011).</li><li>Lim, C. 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=Anthracyclines+induce+calpain-dependent+titin+proteolysis+and+necrosis+in+cardiomyocytes.">Anthracyclines induce calpain-dependent titin proteolysis and necrosis in cardiomyocytes.</a> Journal Biology Chemistry. 279 (9), 8290-8299 (2004).</li><li>Woulfe, K. 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=A+Novel+Method+of+Isolating+Myofibrils+From+Primary+Cardiomyocyte+Culture+Suitable+for+Myofibril+Mechanical+Study.">A Novel Method of Isolating Myofibrils From Primary Cardiomyocyte Culture Suitable for Myofibril Mechanical Study.</a> Frontiers Cardiovascular Medicine. 6, 12 (2019).</li><li>Ait Mou, Y., Bollensdorff, C., Cazorla, O., Magdi, Y., de Tombe, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+cardiac+biophysical+properties.">Exploring cardiac biophysical properties.</a> Global Cardiology Science Practice. 2015, 10 (2015).</li></ol>

저자는 이해상충이 없습니다.

피부 심근세포체를 사용하여 심장 기능의 체외 평가는 생리학적(예를 들어, 스트레치) 및 병리학적 문맥(예를 들어, 허혈)에서 심근세포 수준에서 발생하는 수정을 명확히 하는 중요한 기술을 나타냅니다. 이 방법론은 해동 된 샘플에서 얻은 심근세포에서 기능을 평가하기 위해 최소한의 심근을 요구하는 것과 같은 몇 가지 장점이 있습니다. 광범위한 종(마우스13,쥐1,14,15,토끼16,돼지17,개18,기니피그19 및 인간20)으로부터심근세포및 상이한 심장 위치를 사용하여 아리아, 좌우 심실 또는 경색 심장의 특정 부위를 포함한다. 또한 이 기술은 기본 구성에서 규제 및 수축 구조의 기능을 측정하는 동시에 Ca2+ 및 에너지(ATP)의 특정 농도를 제공할 수 있습니다.
이 기술의 단순성에도 불구하고 몇 가지 중요한 단계가 있습니다. 샘플 수집을 포함하여 처음부터 각 단계의 품질을 보장하는 것이 필수적입니다. 미오필라멘트 단백질은프로테아제(21)에취약하다. 따라서 수집 직후 액체 질소에 샘플을 보관해야 합니다. 이전에 동결되지 않은 신선한 샘플은 훨씬 더 높은 힘을 개발하므로 동일한 프로토콜에서 신선하고 냉동된 샘플에서 수행되는 측정을 혼합하는 것이 좋습니다. 두 번째로 가장 중요한 단계는 심근세포의 추출입니다. 이 절차 동안 대부분의 경우 얼음 샘플을 유지하는 것이 중요합니다. 프로테아제 억제제 칵테일은추출/투과화(22)동안 단백질 분해의 위험을 감소시키는 데 사용될 수 있다. 셋째, 이 단계를 무시했을 때 품질 이내 세포가 감소했다고 지적했기 때문에 정확한 메스 움직임을 사용하여 샘플을 작은 조각으로 절단해야 합니다. 또 다른 중요한 단계는 트리톤을 씻어내고 (세포를 퍼미컬화하지만 그 접착을 촉진) 가능한 한 많은 세포를 유지하는 것 사이의 적절한 균형을 갖기 어렵기 때문에 심근세포를 세척하는 것입니다. 먼저 각 샘플, 종 또는 프로토콜에 대한 추출 및 세척 수를 시도하는 것이 중요합니다. 예를 들어, 우리의 손에, 우리는 ZSF1 비만 쥐 조직 추출은 "지방" 양상을 가지고 있음을 지적, 접착제 동안 이러한 세포를 더 미끄러운 만든 하지만 측정하기 더 어렵지 않다. 우리가 이 문제를 우회하는 방법은 동물 당 세포의 합리적인 수를 가지고 더 많은 실험을 수행하여이었다. 또한, 접착제좋은 세포를 선택하는 것이 중요합니다, 즉 좋은 줄무늬와 합리적인 길이. 심근 세포에는 이러한 기능이 없는 경우 주로 바늘 끝에서 분리되거나 힘이 없음을 일으킵니다. 또한 세포를 바늘에 붙이는 접착제 의 시간과 효능을 고려하여 심근세포 부착에 올바른 접착제를 사용하는 것도 중요합니다. 우리의 손에, 실리콘 접착제(재료의 테이블)빠른 (10-15 분) 충분히 강한 치료. 마지막으로, 마지막 중요한 단계는 세포를 붙인 후 5 분 심근세포를 조심스럽게 들어 올리고 (커버 슬립에 세포를 붙이는 것을 피하기 위해) 우물로 이동하기 전에 (현미경 단계에 의해 끌리는 세포를 피하기 위해)와 관련이 있습니다. 표 7은 이 기술과 관련된 문제 해결, 그 근본 원인 및 빈번한 문제를 극복할 수 있는 가능한 해결 방법을 요약합니다.
이 방법의 주요 제한은 myofilaments활성화 /비활성화 얼마나 빨리 와 같은 myofilament 수축과 관련된 모든 질문에 대답 할 수 없다는 것입니다. 생체 내 설정에서 막 탈극성, 세포내 Ca2+ 증가 및 근안세포에 대한 확산은 심근세포가 수축하는 반면, 피부심근세포Ca2+ 확산에서 근막에 대한 확산은 세포가 Ca2+ 용액에 침수될 때 즉시 발생합니다. Ca2+ 확산의 이 빠른 속도는 근필라멘트 활성화/비활성화분석(23)을편향시킬 것이다.
이러한 실험은 온도, 용액 pH, 기계적 변속(느슨한 재스트레칭 대 느슨함) 및 세포 부착 절차(핀 넥타이 대 접착제)를 포함한 다양한 요인에 의해 영향을받습니다.
기술의 미래 진행은 투과성 심근세포보다는 그대로 기능적 연구를 수행하는 것을 포함한다. 이 기술은 심근세포가 갓 분리된 것에 의존하는 단점이 있습니다(이전에는 동결되지 않음). 이 방법론과 직접적인 관련이 없는 또 다른 중요한 문제는 샘플 냉동 저장의 최대 기간과 관련이 있는 데 큰 영향을 미칠 수 있습니다. 구체적으로, 저장 시간 전반에 걸쳐 근안의 분해 정도를 확립하는 것이 필수적이다(즉, 추출된 심근세포로부터 파생된 양질의 기능데이터를 보장하기 위해 냉동 시료를 얼마나 오래 저장할 수 있는지).

전통적으로 심근 기계적 특성에 대한 평가는 주로 유두 근육 및 trabeculae1,2와같은 다세포제제에서시도되었습니다. 다세포 심장 근육 스트립은 방향 및 힘 생성의 알 수없는 패턴을 가진 수축 심근 세포를 포함하는 세포의 이질적인 인구, 전기 활동 및 스트레스 / 변형 분포뿐만 아니라 주변 결합 조직 매트릭스3,4을포함한다. 콜라겐없이 준비하고 단일 심근 세포를 포함하는 것은 매우 정확하고 제어 된 방식으로 sarcomere 길이와 크로스 브리지 수축 특성의 측정을 허용할것이다5,6. 따라서, 지난 4년 동안, 단일 심근세포6,7의기계적, 수축 및 이완 특성을 조사할 수 있도록 여러 가지 방법론이 개발되었다. 이들 세포의 수축 기능은 사코메 길이 및 크로스 브리지 사이클링 운동학3에크게 의존한다. 따라서, 단 하나 고립 된 심장 세포에서 근육 기능을 직접 조사하는 것이 바람직하다, 그것은 사메레 길이 및 성능뿐만 아니라 크로스 브리지 기능 및 수축 특성을 평가 할 수 있다는 점을 고려. 그러나, μN 수준에서 힘 측정을 기록하는 동안 합리적인 광학 sarcomere 해상도로 기능성 심근세포를 분리하고 부착하는 것은 여전히 도전적이고진화3,6이다. 다른 과제는 갓 수거된 생검에서 심근세포를 분리하기 위해 설치해야 하는 물류입니다. 인간 생검 수집의 예측 불가능성은, 예를 들면, 실험의 타당성을 위태롭게 할 수 있습니다.
더욱이, 과학적 절차에 대한 동물 실험의 교체, 감소 및 정제에 관한 윤리적 관심사 (3R의 원칙)는 세포 및 조직 수준, 바람직하게는 인간의 생검 또는 작은 동물 샘플에서 연구 변화를 촉진했다. 실제로 복잡성의 작은 수준에서 시험관 내 심장 기능을 평가하는 방법론의 점진적 개선은 전신에 결과의 적절한 통합을 허용하고 임상 시나리오7로변환할 수 있습니다. 전부, 심근구를 추출하기 위해 -80°C에 저장된 샘플을 사용하는 것이 매력적인 대안일 수 있다.
심근 조직은 작은 조각으로 절단하고 박격포와 봉제와 균질화된다. 이러한 균질화의 결과는 다양한 정도의 사콜렘말 손상을 가진 피부 뭉닝 및 분리된 세포의 현탁액으로, 이 때 근근증이 입욕 매체에 노출되고 모든 세포 성분이 씻겨나난다. 사콜마에서 멀리 떨어져 있는 근시릴과 같은 구조물은 보존되어 있습니다. 따라서, 근시릴라 장치와 관련된 사컴레 단축 및 기능성 특성은 그대로 유지되며8,9를기록할 수 있다.
심근세포 력 측정 시스템은 심근세포 길이를 조정하는 데 사용되는 전자기 모터와 동급성 심근세포 수축을 측정하는 힘 트랜스듀서로 구성됩니다. 투과, 또는 피부, 심근세포는 편안한 용액 ([Ca2+] < 10 nM)을 포함하는 실험 챔버에 배치되고 실리콘 접착2 얇은 바늘 : 하나는 모터에 부착되고 다른 하나는 힘 트랜스듀서에 부착됩니다. 광학 시스템은 심근세포 형태와 사코프레 길이를 결정하는 데 사용됩니다. 실험 프로토콜은 종종 상이한 Ca2+ 농도를 포함하는 완충액의 일련의 힘 기록, 액틴-미오신 크로스 브리지 운동제의 결정 및 미리 정의된 sarcomere 길이에서 장착된 심근세포의 수동 장력측정(그림 1)으로구성된다. 액체 질소에서 동결된 심근 샘플로부터 의 분리(그리고 이후 -80°C에 저장됨)는 단단 면적당 최대 Ca2+활성화(활성) 힘을 측정하기 위한 세포 역학 및 단백질 생화학을 활용하는 기술입니다(T 활성, kN∙m-2), Ca 2+ 독립(T 활성, kN+m-2), Ca 2+-독립(T 활성, kN+m-2), Ca 2+ 독립(T 활성, kN+m-2), Ca 2+ 독립(T활성,kN+m-2), Ca2+독립(패시브) 장력(T패시브,kN+m-2), 근안회카2+민감도(pCa50),근안회(nHill), 힘 재개발(ktr) 및 T활성,T패시브,pCa50,nHill 및 ktr의 사코머 길이 종속성.
이 프로토콜의 목표는 다른 종에서 냉동 된 샘플에서 분리 된 단일 피부 심근 세포의 기능적 기계적 특성을 평가하는 신뢰할 수있는 절차로 심근 세포 력 측정 시스템의 잠재력을 설명하고 요약하는 것입니다.

<table><tbody><tr><td>Acetone</td><td>Sigma</td><td>34580</td><td></td></tr><tr><td>Adenosine 5&amp;rsquo;-triphosphate disodium salt hydrate (Na2ATP)</td><td>Sigma</td><td>A2383</td><td></td></tr><tr><td>Calcium carbonate (CaCO3)</td><td>Merck</td><td>1.02067.0500</td><td></td></tr><tr><td>Imidazole</td><td>VWR</td><td>24720.157</td><td></td></tr><tr><td>Magnesium chloride hexahydrate (MgCl2.6H2O)</td><td>Merck</td><td>1.05833.0250</td><td></td></tr><tr><td>Magnesium chloride solution (MgCl2 1M)</td><td>Sigma</td><td>M1028</td><td></td></tr><tr><td>N,N-Bis(2-hydroxyethyl)taurine (BES)</td><td>Sigma</td><td>B9879</td><td></td></tr><tr><td>Phosphocreatine dissodium salt hydrate (Na2PCr)</td><td>Sigma</td><td>P7936</td><td></td></tr><tr><td>Potassium chloride (KCl)</td><td>Merck</td><td>1.04936.1000</td><td></td></tr><tr><td>Potassium hydroxide (KOH)</td><td>Merck</td><td>8.14353.1000</td><td></td></tr><tr><td>Propionic acid (C3H6O2)</td><td>Merck</td><td>8.00605.0500</td><td></td></tr><tr><td>Silicone Squeeze Tube</td><td>Marineland</td><td>31003</td><td></td></tr><tr><td>Tritiplex (EGTA)</td><td>Merck</td><td>1.08435.0025</td><td></td></tr><tr><td>Triton&amp;reg; X-100 10%</td><td>Merck</td><td>648463</td><td></td></tr><tr><td>Tissue homogeneizer (GKH GT Motor Control)</td><td>Terre Haute Glas-col</td><td></td><td></td></tr><tr><td>Length Controller (Model 315C-I)</td><td>Aurora Scientific</td><td></td><td></td></tr><tr><td>Force Transducer (Model 403 A)</td><td>Aurora Scientific</td><td></td><td></td></tr><tr><td>Software ASI 600A</td><td>Aurora Scientific</td><td></td><td></td></tr><tr><td>Sotware VSL (Model 900B)</td><td>Aurora Scientific</td><td></td><td></td></tr><tr><td>Inverted Microscope (IX51)</td><td>Olympus</td><td></td><td></td></tr></tbody></table>

in vitro assessment of cardiac function using skinned cardiomyocytes

이 기사에서는 단일 과구("skinned") 심근세포를 분리하고 기능적 연구를 수행하기 위해 힘 측정 장치 및 모터에 부착하는 데 필요한 단계를 설명합니다. 이러한 연구는 심근세포 강성(수동력)과 다른 칼슘(Ca2+)함유 솔루션으로 활성화하여 최대 력 개발, 근안타멘트 Ca2+-감도(pCa50),쿠퍼티움(nHill) 및 힘 재개발 속도(ktr)를 측정할 수 있습니다. 이 방법은 또한 근막에 직접 작용하는 약물의 효과와 심근세포의 활성 및 수동 적 특성모두에 외인성 재조합 단백질의 발현을 가능하게합니다. 임상적으로, 피부 심근 세포 연구는 많은 심근 질환의 병리생리학을 강조하고 myofilaments를 대상으로 치료 개입의 영향의 체외 평가를 허용. 전부, 이 기술은 열린 심혼 또는 이식 수술 도중 얻은 동물 모형 및 인간 조직에서 시험관 내 및 생체 내 파라미터 사이 상관관계를 조사하여 심장 병리생리학의 설명을 가능하게 합니다.

Watch this Scientific Journal Video about In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes at JoVE.com

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

이 프로토콜은 피부 심장 근구를 사용하여 심장 기능의 추출 및 평가 기술을 단계별로 설명하는 것을 목표로합니다. 이 방법론은 마우스에서 남성에 이르기까지 다른 심장 위치에서 수집 할 수있는 작은 냉동 생검을 사용하여 myofilament 기능의 측정 및 급성 변조를 허용합니다.

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 ...

in-vitro-assessment-of-cardiac-function-using-skinned-cardiomyocytes

Research

JoVE Journal

Medicine

6.1K 조회수.  Universidade do Porto. 이 프로토콜은 피부 심장 근구를 사용하여 심장 기능의 추출 및 평가 기술을 단계별로 설명하는 것을 목표로합니다. 이 방법론은 마우스에서 남성에 이르기까지 다른 심장 위치에서 수집 할 수있는 작은 냉동 생검을 사용하여 myofilament 기능의 측정 및 급성 변조를 허용합니다.

비디오: In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

연구

생체공학

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated researchers in the stem cell and bioengineering fields to develop in vitro high-fidelity human myocardium for both basic research and clinical applications. However, the immature phenotype of cardiac cells is a limitation on obtaining tissues that functionally mimic the adult myocardium, which is mainly characterized by mechanical and electrical signals. Thus, the purpose of this protocol is to prepare and mature the target cell population through electromechanical stimulation, recapitulating physiological parameters. Cardiac tissue engineering is evolving toward more biological approaches, and strategies based on biophysical stimuli, thus, are gaining momentum. The device developed for this purpose is unique and allows individual or simultaneous electrical and mechanical stimulation, carefully characterized and validated. In addition, although the methodology has been optimized for this stimulator and a specific cell population, it can easily be adapted to other devices and cell lines. The results here offer evidence of the increased cardiac commitment of the cell population after electromechanical stimulation. Electromechanically stimulated cells show an increased expression of main cardiac markers, including early, structural, and calcium-regulating genes. This cell conditioning could be useful for further regenerative cell therapy, disease modeling, and high-throughput drug screening.

Here we present a protocol for training a cell population using electrical and mechanical stimuli emulating cardiac physiology. This electromechanical stimulation enhances the cardiomyogenic potential of the treated cells and is a promising strategy for further cell therapy, disease modeling, and drug screening.

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated ...

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.

This protocol describes the procedure for sectioning and culturing human cardiac slices for preclinical drug testing and details the use of optical mapping for recording transmembrane voltage and intracellular calcium signals simultaneously from these slices.

인간 조직형 심장 슬라이스의 이중 전압 및 칼슘 광학 매핑에 의한 전임상 심장 전극생리학 평가

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

인간 iPSC 유래 심근세포, 심장 섬유아세포 및 내피 세포를 사용하여 3D 심장 미세 조직 배열의 제조

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...

Cardiac Spheroids as in vitro Bioengineered Heart Tissues to Study Human Heart Pathophysiology

Despite several advances in cardiac tissue engineering, one of the major challenges to overcome remains the generation of a fully functional vascular network comprising several levels of complexity to provide oxygen and nutrients within bioengineered heart tissues. Our laboratory has developed a three-dimensional in vitro model of the human heart, known as the "cardiac spheroid" or "CS". This presents biochemical, physiological, and pharmacological features typical of the human heart and is generated by co-culturing its three major cell types, such as cardiac myocytes, endothelial cells, and fibroblasts. Human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs or iCMs) are co-cultured at ratios approximating the ones found in vivo with human cardiac fibroblasts (HCFs) and human coronary artery endothelial cells (HCAECs) in hanging drop culture plates for three to four days. The confocal analysis of CSs stained with antibodies against cardiac Troponin T, CD31 and vimentin (markers for cardiac myocytes, endothelial cells and fibroblasts, respectively) shows that CSs present a complex endothelial cell network, resembling the native one found in the human heart. This is confirmed by the 3D rendering analysis of these confocal images. CSs also present extracellular matrix (ECM) proteins typical of the human heart, such as collagen type IV, laminin and fibronectin. Finally, CSs present a contractile activity measured as syncytial contractility closer to the one typical of the human heart compared to CSs that contain iCMs only. When treated with a cardiotoxic anti-cancer agent, such as doxorubicin (DOX, used to treat leukemia, lymphoma and breast cancer), the viability of DOX-treated CSs is significantly reduced at 10 &#181;M genetic and chemical inhibition of endothelial nitric oxide synthase, a downstream target of DOX in HCFs and HCAECs, reduced its toxicity in CSs. Given these unique features, CSs are currently used as in vitro models to study heart biochemistry, pathophysiology, and pharmacology.

This protocol aims to fabricate 3D cardiac spheroids (CSs) by co-culturing cells in hanging drops.&#160;Collagen-embedded CSs are treated with doxorubicin (DOX, a cardiotoxic agent) at physiological concentrations to model heart failure. In vitro testing using DOX-treated CSs may be used to identify novel therapies for heart failure patients.

인간 심장 병리학을 연구하기 위하여 체외 생체 공학심장 조직에서와 같이 심장 스페로이드

Despite several advances in cardiac tissue engineering, one of the major challenges to overcome remains the generation of a fully functional vascular ...

Hybrid Cell Analysis System to Assess Structural and Contractile Changes of Human iPSC-Derived Cardiomyocytes for Preclinical Cardiac Risk Evaluation

Cardiac contractility assessment is of immense importance for the development of new therapeutics and their safe transition into clinical stages. While human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold promise to serve as a human-relevant model in preclinical phases of drug discovery and safety pharmacology, their maturity is still controversial in the scientific community and under constant development. We present a hybrid contractility and impedance/extracellular field potential (EFP) technology, adding significant pro-maturation features to an industry-standard 96-well platform.
The impedance/EFP system monitors cellular functionality in real-time. Besides the beat rate of contractile cells, the electrical impedance spectroscopy readouts detect compound-induced morphological changes like cell density and integrity of the cellular monolayer. In the other component of the hybrid cell analysis system, the cells are cultured on bio-compliant membranes that mimic the mechanical environment of real heart tissue. This physiological environment supports the maturation of hiPSC-CMs in vitro, leading to more adult-like contractile responses including positive inotropic effects after treatment with isoproterenol, S-Bay K8644, or omecamtiv mecarbil. Parameters such as the amplitude of contraction force (mN/mm2) and beat duration also reveal downstream effects of compounds with influence on electrophysiological properties and calcium handling.
The hybrid system provides the ideal tool for holistic cell analysis, allowing preclinical cardiac risk assessment beyond the current perspectives of human-relevant cell-based assays.

The analysis of changes in contractile function and cellular integrity of human iPSC-derived cardiomyocytes is of immense importance for nonclinical drug development. A hybrid 96-well cell analysis system addresses both parameters in a real-time and physiological manner for reliable, human-relevant results, necessary for a safe transition into clinical stages.

전임상 심장 위험 평가를 위해 인간 iPSC 유래 심근 세포의 구조적 및 수축성 변화를 평가하기위한 하이브리드 세포 분석 시스템

Cardiac contractility assessment is of immense importance for the development of new therapeutics and their safe transition into clinical stages. While ...

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used in regulatory submissions. Here, we extend their use to cardiac medical device safety or performance assessments. We developed a novel method to evaluate cardiac medical device contractile properties in robustly contracting 2D hiPSC-CMs monolayers plated on a flexible extracellular matrix (ECM)-based hydrogel substrate. This tool enables the quantification of the effects of cardiac electrophysiology device signals on human cardiac function (e.g., contractile properties) with standard laboratory equipment. The 2D hiPSC-CM monolayers were cultured for 2-4 days on a flexible hydrogel substrate in a 48-well format.
The hiPSC-CMs were exposed to standard cardiac contractility modulation (CCM) medical device electrical signals and compared to control (i.e., pacing only) hiPSC-CMs. The baseline contractile properties of the 2D hiPSC-CMs were quantified by video-based detection analysis based on pixel displacement. The CCM-stimulated 2D hiPSC-CMs plated on the flexible hydrogel substrate displayed significantly enhanced contractile properties relative to baseline (i.e., before CCM stimulation), including an increased peak contraction amplitude and accelerated contraction and relaxation kinetics. Furthermore, the utilization of the flexible hydrogel substrate enables the multiplexing of the video-based cardiac-excitation contraction coupling readouts (i.e., electrophysiology, calcium handling, and contraction) in healthy and diseased hiPSC-CMs. The accurate detection and quantification of the effects of cardiac electrophysiological signals on human cardiac contraction is vital for cardiac medical device development, optimization, and de-risking. This method enables the robust visualization and quantification of the contractile properties of the cardiac syncytium, which should be valuable for nonclinical cardiac medical device safety or effectiveness testing. This paper describes, in detail, the methodology to generate 2D hiPSC-CM hydrogel substrate monolayers.

Here, we demonstrate a non-invasive cardiac medical device contractility evaluation method using 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, plated on a flexible substrate, coupled with video-based microscopy. This tool will be useful for the in vitro evaluation of the contractile properties of cardiac electrophysiology devices.

2D 인간 줄기 세포 유래 심근 세포에서 심장 수축성 조절 요법의 평가

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used ...

High-Throughput Cardiotoxicity Screening Using Mature Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Monolayers

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical cardiotoxicity testing. In two-dimensional monolayer formats, hiPSC-CMs recapitulate the structure and function of the adult human heart muscle cells when cultured on an optimal extracellular matrix (ECM). A human perinatal stem cell-derived ECM (maturation-inducing extracellular matrix-MECM) matures the hiPSC-CM structure, function, and metabolic state in 7 days after plating. 
Mature hiPSC-CM monolayers also respond as expected to clinically relevant medications, with a known risk of causing arrhythmias and cardiotoxicity. The maturation of hiPSC-CM monolayers was an obstacle to the widespread adoption of these valuable cells for regulatory science and safety screening, until now. This article presents validated methods for the plating, maturation, and high-throughput, functional phenotyping of hiPSC-CM electrophysiological and contractile function. These methods apply to commercially available purified cardiomyocytes, as well as stem cell-derived cardiomyocytes generated in-house using highly efficient, chamber-specific differentiation protocols. 
High-throughput electrophysiological function is measured using either voltage-sensitive dyes (VSDs; emission: 488 nm), calcium-sensitive fluorophores (CSFs), or genetically encoded calcium sensors (GCaMP6). A high-throughput optical mapping device is used for optical recordings of each functional parameter, and custom dedicated software is used for electrophysiological data analysis. MECM protocols are applied for medication screening using a positive inotrope (isoprenaline) and human Ether-a-go-go-related gene (hERG) channel-specific blockers. These resources will enable other investigators to successfully utilize mature hiPSC-CMs for high-throughput, preclinical cardiotoxicity screening, cardiac medication efficacy testing, and cardiovascular research.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer an alternative to using animals for preclinical cardiotoxicity screening. A limitation to the widespread adoption of hiPSC-CMs in preclinical toxicity screening is the immature, fetal-like phenotype of the cells. Presented here are protocols for robust and rapid maturation of hiPSC-CMs.

성숙한 인간 유도 만능 줄기 세포 유래 심근 세포 단층을 사용한 고처리량 심장 독성 스크리닝

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical ...

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

마우스 심근의 분리 및 생리 학적 분석

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

생물학

Model of Ischemic Heart Disease and Video-Based Comparison of Cardiomyocyte Contraction Using hiPSC-Derived Cardiomyocytes

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with small-animal models such as rodents. However, the physiology of the human heart differs significantly from that of the rodent heart, underscoring the need for clinically relevant models to study heart disease. Here, we present a protocol to model ischemic heart disease using cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS-CMs) and to quantify the damage and functional impairment of the ischemic cardiomyocytes. Exposure to 2% oxygen without glucose and serum increases the percentage of injured cells, which is indicated by staining of the nucleus with propidium iodide, and decreases cellular viability. These conditions also decrease the contractility of hiPS-CMs as confirmed by displacement vector field analysis of microscopic video images. This protocol may furthermore provide a convenient method for personalized drug screening by facilitating the use of hiPS cells from individual patients. Therefore, this model of ischemic heart disease, based on iPS-CMs of human origin, can provide a useful platform for drug screening and further research on ischemic heart disease.

We present a model of ischemic heart disease using cardiomyocytes derived from human induced pluripotent stem cells, together with a method for quantitative evaluation of tissue damage caused by ischemia. This model can provide a useful platform for drug screening and further research on ischemic heart disease.