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>
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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetone</td>
			<td>Sigma</td>
			<td>34580</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5&#8217;-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&#174; 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

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JoVE Journal

Medicine

5.9K 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

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of cardiovascular disease. Although molecular biology is necessary in identifying key changes in the signaling pathway, it is not a surrogate for functional significance. While physiology can provide answers to the question of function, combining physiology with biochemical assessment of metabolites in the intact, beating heart allows for a complete picture of cardiac function and energetics. For years, our laboratory has utilized isolated heart perfusions combined with nuclear magnetic resonance (NMR) spectroscopy to accomplish this task. Left ventricular function is assessed by Langendorff-mode isolated heart perfusions while cardiac energetics is measured by performing 31P magnetic resonance spectroscopy of the perfused hearts. With these techniques, indices of cardiac function in combination with levels of phosphocreatine and ATP can be measured simultaneously in beating hearts. Furthermore, these parameters can be monitored while physiologic or pathologic stressors are instituted. For example, ischemia/reperfusion or high workload challenge protocols can be adopted. The use of aortic banding or other models of cardiac pathology are apt as well. Regardless of the variants within the protocol, the functional and energetic significance of molecular modifications of transgenic mouse models can be adequately described, leading to new insights into the associated enzymatic and metabolic pathways. Therefore, 31P NMR spectroscopy in the isolated perfused heart is a valuable research technique in animal models of cardiovascular disease.

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Langendorff-mode isolated heart perfusion, in conjunction with 31P NMR spectroscopy, combines the fields of biochemistry and physiology into one experiment. The protocol allows for the dynamic measurement of high energy phosphate content and turnover in the heart while concurrently monitoring physiologic function. When performed correctly, this is a valuable technique in the assessment of cardiac energetics.

를 사용하여 절연 마우스 마음에 심장 기능 및 에너 지학 평가 31 P NMR 분광학

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of ...

연구

의학

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery (SALLI) MRI in Rats

Small animal magnetic resonance imaging is an important tool to study cardiac function and changes in myocardial tissue. The high heart rates of small animals (200 to 600 beats/min) have previously limited the role of CMR imaging. Small animal Look-Locker inversion recovery (SALLI) is a T1 mapping sequence for small animals to overcome this problem 1. T1 maps provide quantitative information about tissue alterations and contrast agent kinetics. It is also possible to detect diffuse myocardial processes such as interstitial fibrosis or edema 1-6. Furthermore, from a single set of image data, it is possible to examine heart function and myocardial scarring by generating cine and inversion recovery-prepared late gadolinium enhancement-type MR images 1.
	The presented video shows step-by-step the procedures to perform small animal CMR imaging. Here it is presented with a healthy Sprague-Dawley rat, however naturally it can be extended to different cardiac small animal models.

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery SALLI MRI in Rats

Contrast enhanced cardiac magnetic resonance (CMR) imaging allows comprehensive in vivo assessment of the heart in small animal models of cardiovascular disease. Here we detail the procedures to perform CMR imaging, reconstruction and analysis using Small Animal Lock-Locker Inversion Recovery (SALLI) in rats.

작은 동물 룩 로커 반전 복구 (SALLI) 쥐의 MRI를 사용하여 심장 기능 및 심근 형태학의 평가

Small animal magnetic resonance  imaging is an important tool to study cardiac function and changes in myocardial  tissue. The high heart rates of small ...

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

경 흉부 심 초음파에 의한 폐동맥 수축의 마우스 모델에서 마우스 오른쪽 심실 구조 및 기능의 평가

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

Transcutaneous Assessment of Renal Function in Conscious Rodents

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and time-consuming. In addition, serial blood or urine samples are required, with the associated stress for the experimental animals. A recent technique significantly reduces the investment in time and resources, minimizing the invasiveness and the animal stress, but being equally valid as the traditional approaches. The method measures transcutaneously renal function. Using an optical device and the exogenous renal marker fluorescein isothiocyanate (FITC)-sinistrin, this technique is capable of measuring the elimination kinetics of the marker through the skin. With neither blood nor urine samples nor the associated laboratory assays needed, the results of the transcutaneous measurement are almost instantaneously available. The method has been already validated in different species and successfully applied in several models of renal pathology. Moreover, due to its minimally invasive characteristics, it is suitable for sequential measurements within the same animal. Here is provided a detailed protocol to carry out the transcutaneous assessment of renal function in rodents.

Determination of glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional procedures to measure this parameter are cumbersome and require a large investment of time. Here we describe a faster and minimally invasive method to determinate GFR transcutaneously.

의식 설치류에서 신장 기능의 경피 평가

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

준비 및<em&gt; 체외</em&gt; 자화 특성의 내피 세포를 미르 변성

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

인간 지방 조직 Microvascular 함수 Videomicroscopy를 사용 하 여 평가

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...

Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation time and increases the risk of adverse post-transplantation outcomes. Moreover, the static nature of CS does not allow for organ evaluation or intervention during the preservation interval. Normothermic ex situ heart perfusion (ESHP) is a novel method for preservation of the donated heart that minimizes cold ischemia by providing oxygenated, nutrient-rich perfusate to the heart. ESHP has been shown to be non-inferior to CS in the preservation of standard-criteria donor hearts and has also facilitated the clinical transplantation of the hearts donated after the circulatory determination of death. Currently, the only available clinical ESHP device perfuses the heart in an unloaded, non-working state, limiting assessments of myocardial performance. Conversely, ESHP in working mode provides the opportunity for comprehensive evaluation of cardiac performance by assessment of functional and metabolic parameters under physiologic conditions. Moreover, earlier experimental studies have suggested that ESHP in working mode may result in improved functional preservation. Here, we describe the protocol for ex situ perfusion of the heart in a large mammal (porcine) model, which is reproducible for different animal models and heart sizes. The software program in this ESHP apparatus allows for real-time and automated control of the pump speed to maintain desired aortic and left atrial pressure and evaluates a variety of functional and electrophysiological parameters with minimal need for supervision/manipulation.

Normothermic ex situ heart perfusion (ESHP), preserves the heart in a beating, semi-physiologic state. When performed in a working mode, ESHP provides the opportunity to perform sophisticated assessments of donor heart function and organ viability. Here, we describe our method for myocardial performance evaluation during ESHP.

작업 모드에 Situ 심장 관류 Ex normothermic: 심장 기능 및 대사 평가

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation ...

Echocardiographic Assessment of Cardiac Anatomy and Function in Adult Rats

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional imaging to study the cardiac anatomy of the left ventricle and M-mode echo to assess its dimensions. However, this could limit a comprehensive study. Herein, we describe a protocol that allows an assessment of the heart chamber size, left ventricular function (systolic and diastolic) and valvular function. A conventional medical ultrasound machine was used in this protocol and different echo views were obtained through left parasternal, apical and suprasternal windows. In the left parasternal window, the long and short axis were acquired to analyze left chamber dimensions, right ventricle and pulmonary artery dimensions, and mitral, pulmonary and aortic valve function. The apical window allows the measurement of heart chamber dimensions and evaluation of systolic and diastolic parameters. It also allows Doppler assessment with detection and quantification of heart valve disturbances (regurgitation or stenosis). Different segments and walls of the left ventricle are visualized throughout all views. Finally, the ascending aorta, aortic arch, and descending aorta can be imaged through the suprasternal window. A combination of ultrasound imaging, Doppler flow and tissue Doppler assessment have been obtained to study cardiac morphology and function. This represents an important contribution to improve the assessment of cardiac function in adult rats with impact for research using these animal models.

A non-invasive protocol for transthoracic echocardiography assessment of cardiac anatomy and function for adult rats is presented in the current study. The heart valves, all four cardiac chambers and the ascending aorta, aortic arch and descending aorta are studied in detail.

성인 쥐에서 심장 해부학 및 기능의 심초음파 평가

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

이식 품질 평가를 위해 화학 생검 사용

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...

Semi-Minimal Invasive Method to Induce Myocardial Infarction in Rats and the Assessment of Cardiac Function by an Isolated Working Heart System

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily and highly reproducible MI induction procedure is required to obtain further insight and better understanding of the underlying pathological changes. This procedure can also be used to evaluate the effects or potency of new and promising treatments (as drugs or interventions) in acute MI, subsequent remodeling and heart failure (HF). After intubation and pre-operative preparation of the animal, an anesthetic protocol with isoflurane was performed, and the surgical procedure was conducted&#160;quickly. Using a minimally invasive approach, the left anterior descending artery (LAD) was located and occluded by a ligature. The occlusion can be performed acutely for subsequent reperfusion (ischemia/reperfusion injury). Alternatively, the vessel can be ligated permanently to investigate the development of chronic MI, remodeling or HF. Despite common pitfalls, the drop-out rates are minimal. Various treatments such as remote ischemic conditioning can be examined for their cardioprotective potential pre-, peri- and post-operatively. The post-operative recovery was quick as the anesthesia was precisely controlled and the duration of the operation was short. Post-operative analgesia was administered for three days. The minimally invasive procedure reduces the risk of infection and inflammation. Furthermore, it facilitates rapid recovery. The &#8220;working heart&#8221; measurements were performed ex vivo and enabled precise control of preload, afterload and flow. This procedure requires specific equipment and training for adequate performance. This manuscript provides a detailed step-by-step introduction for conducting these measurements.

This article presents an efficient method to perform myocardial ischemia and subsequent chronic reperfusion in rats using a minimally invasive approach. In addition, left ventricular hemodynamic function of rats is assessed by echocardiography and isolated working heart methods.&#160;

쥐의 심근 경색과 격리 된 작업 심장 시스템에 의한 심장 기능 평가를 유도하는 반 최소 침습 적 방법

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily ...