Name: assessment of sarcoplasmic reticulum calcium reserve and intracellular diastolic calcium removal in isolated ventricular cardiomyocytes
Uploaded: 2017-09-18T13:00:00
Description: Watch this Scientific Journal Video about Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes at JoVE.com

这项工作得到了中国国家自然科学基金 (No. 81100159, 东吴莱; 81401147, 橘红章), 浙江省医疗卫生科学项目 (No. 201646246, 东吴赖) 的资助, 以及卫生科学和杭州市技术方案 (No. 2013A28, 丁林)。

所有动物实验都是按照中国医学科学院和浙江大学实验研究中心机构动物保育与使用委员会批准的协议进行的. 
 1. 解决方案准备 <ol> <li> 准备 表 1 . </li> 中所述的所有解决方案
 2. 分离左心室 (lv) 心肌细胞 注意: LV 心肌细胞被隔离酶使用 Langendroff 灌注系统, 如前所述 7 , 8 <sup...

<ol>
	<li>Capel, R. A., Terrar, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+Ca+(2+)-dependent+mechanisms+for+the+initiation+of+the+heartbeat.">The importance of Ca (2+)-dependent mechanisms for the initiation of the heartbeat.</a> Front Physiol. 6, 80 (2015).</li><li>Bers, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+cycling+and+signaling+in+cardiac+myocytes.">Calcium cycling and signaling in cardiac myocytes.</a> Annu Rev Physiol. 70, 23-49 (2008).</li><li>Zhao, S. M., Wang, Y. L., Guo, C. Y., Chen, J. L., Wu, Y. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+decay+of+Ca2++homeostasis+in+the+development+of+diabetic+cardiomyopathy.">Progressive decay of Ca2+ homeostasis in the development of diabetic cardiomyopathy.</a> Cardiovasc Diabetol. 13, 75 (2014).</li><li>Pereira, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signaling+in+diabetic+cardiomyocytes.">Calcium signaling in diabetic cardiomyocytes.</a> Cell calcium. 56 (5), 372-380 (2014).</li><li>Coppini, 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=Late+sodium+current+inhibition+reverses+electromechanical+dysfunction+in+human+hypertrophic+cardiomyopathy.">Late sodium current inhibition reverses electromechanical dysfunction in human hypertrophic cardiomyopathy.</a> Circulation. 127 (5), 575-584 (2013).</li><li>Ferrantini, 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=R4496C+RyR2+mutation+impairs+atrial+and+ventricular+contractility.">R4496C RyR2 mutation impairs atrial and ventricular contractility.</a> J Gen Physiol. 147 (1), 39-52 (2016).</li><li>Li, L., Chu, G., Kranias, E. G., Bers, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+myocyte+calcium+transport+in+phospholamban+knockout+mouse+relaxation+and+endogenous+CaMKII+effects.">Cardiac myocyte calcium transport in phospholamban knockout mouse relaxation and endogenous CaMKII effects.</a> Am J Physiol. 274, H1335-H1347 (1998).</li><li>Cheng, 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=CaMKII+inhibition+in+heart+failure,+beneficial,+harmful,+or+both.">CaMKII inhibition in heart failure, beneficial, harmful, or both.</a> Am J Physiol Heart Circ Physiol. 302, H1454-H1465 (2012).</li><li>Lai, 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=The+Rho+kinase+inhibitor,+fasudil,+ameliorates+diabetes-induced+cardiac+dysfunction+by+improving+calcium+clearance+and+actin+remodeling.">The Rho kinase inhibitor, fasudil, ameliorates diabetes-induced cardiac dysfunction by improving calcium clearance and actin remodeling.</a> J Mol Med (Berl). 95 (2), 155-165 (2017).</li><li>Lai, 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=Stretch+Current-Induced+Abnormal+Impulses+in+CaMKII&#948;+Knockout+Mouse+Ventricular+Myocytes.">Stretch Current-Induced Abnormal Impulses in CaMKII&#948; Knockout Mouse Ventricular Myocytes.</a> J Cardiovasc Electrophysiol. 24 (4), 457-463 (2013).</li><li>Xu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+of+L-type+calcium+current+and+cardiac+function+in+CaMKII{delta}+knockout+mice.">Alterations of L-type calcium current and cardiac function in CaMKII{delta} knockout mice.</a> Circ Res. 107 (3), 398-407 (2010).</li><li>Roussel, 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=Palmitoyl-carnitine+increases+RyR2+oxidation+and+sarcoplasmic+reticulum+Ca2++leak+in+cardiomyocytes:+Role+of+adenine+nucleotide+translocase.">Palmitoyl-carnitine increases RyR2 oxidation and sarcoplasmic reticulum Ca2+ leak in cardiomyocytes: Role of adenine nucleotide translocase.</a> Biochim Biophys Acta. 1852 (5), 749-758 (2015).</li><li>Yaras, 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=Effects+of+diabetes+on+ryanodine+receptor+Ca2++release+channel+(RyR2)+and+Ca2++homeostasis+in+rat+heart.">Effects of diabetes on ryanodine receptor Ca2+ release channel (RyR2) and Ca2+ homeostasis in rat heart.</a> Diabetes. 54 (11), 3082-3088 (2005).</li><li>Hu, Y., 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=Adenovirus-Mediated+Overexpression+of+O-GlcNAcase+Improves+Contractile+Function+in+the+Diabetic+Heart.">Adenovirus-Mediated Overexpression of O-GlcNAcase Improves Contractile Function in the Diabetic Heart.</a> Circ Res. 96 (9), 1006-1013 (2005).</li></ol>

从 SR 释放的钙通量是心脏收缩的主要的 Ca2 +来源。在某种程度上, sr ca2 +内容的振幅和从锶中释放出来的分数 ca2 +反映了 sr ca2 +保留可用于心脏收缩。另一方面, ca2 +保留的 sr 依赖于 sr ca2 +再的能力、ca2 +泄漏的 sr, 以及它们在舒张期间的 sr、12、13、14之间的平衡。在我?...

细胞内钙 ([Ca2 +]i) 回收在心肌细胞收缩和舒张功能调节中起着关键作用1。正如我们所知道的, 钙诱导 Ca2 +释放启动的励磁收缩耦合, 这将电信号转换为收缩。膜去极化激活心肌 L 型 ca2 +通道, 通过兰尼受体 2 (RyR2) 诱导从 SR 到细胞质的 ca2 +释放。瞬时升高的细胞质 Ca2 +启动收缩的纤维。在舒张期间, 细胞质钙2 +通过 sr Ca2 +atp 酶 2 (SERCA2) reuptaken 进入 sr, 并通过 Na+-Ca2 +交换器 (NCX)2从心肌细胞中抽出。这个过程导致收缩-松弛循环在心肌细胞。
心脏 SR 是一个细胞内膜网络周围的收缩机械。它作为一个 ca2 +的收缩库, 它吸收细胞内 ca2 +在放松。可用于节拍的 SR Ca2 +准备金是确定心脏收缩力的方法。同时, 移除细胞内钙2 +对于心脏舒张是至关重要的。在一些病理生理学条件下, 如糖尿病和心力衰竭, 受损的 ca2 +清除和抑郁症的 SR ca2 +存储在心肌细胞可能涉及心脏功能障碍的过程2,3 ,4。
对于在心肌细胞中测量 SR ca2 +释放和舒张期 ca2 + , 有两种广泛使用的方法: 基于膜片钳5、6和咖啡因诱导的 ca 的 NCX 电流的完整性2 +脉冲基于 Ca2 +荧光成像7,8,9。前者的方法取决于这样一个事实: 从 SR 中释放的 Ca2 +主要是通过 NCX 从单元格中抽取出来的。然而, 这种方法是有限的, 其要求先进的设备和熟练的操作。在本研究中, 我们描述了一种简便的方法来评估 SR ca2 +储备和 ca2 +去除在心肌细胞通过测量咖啡因诱导的 ca2 +脉冲的基础上的 ca2 +荧光成像系统。简单地说, 胞内 Ca2 +荧光由 Fura-2 表示。通过对刺激系统和灌注系统的相互关联, 提出了一种自动切换灌注和起搏系统的方案。10毫米咖啡因被用来迅速诱导总 Ca2 +释放在 SR。从单指数曲线拟合得到了钙瞬变和咖啡因诱导的钙离子脉冲的指数衰减时间常数 (Tau), 反映了 SERCA 和 NCX 对舒张 Ca 的贡献2 + 。

<table>
	<tbody>
		<tr>
			<td>NaCl</td>
			<td>Alfa Aesar</td>
			<td>E31K43</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Alfa Aesar</td>
			<td>I02T070</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Alfa Aesar</td>
			<td>G22u018</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPEs</td>
			<td>Sigma</td>
			<td>SLBM 7880V</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Alfa Aesar</td>
			<td>10189341</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Alfa Aesar</td>
			<td>10154048</td>
			<td></td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Alfa Aesar</td>
			<td>10144B17</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Alfa Aesar</td>
			<td>F21S033</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Alfa Aesar</td>
			<td>C31U038</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamic</td>
			<td>Alfa Aesar</td>
			<td>10149849</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Alfa Aesar</td>
			<td>J5407a</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>SLBM6826V</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase A</td>
			<td>Roche</td>
			<td>10103586001</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type II</td>
			<td>Worthington</td>
			<td>45k16005</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Roche</td>
			<td>735094</td>
			<td></td>
		</tr>
		<tr>
			<td>caffeine</td>
			<td>Sigma</td>
			<td>C0750</td>
			<td></td>
		</tr>
		<tr>
			<td>Fura-2 AM</td>
			<td>Invitrogen</td>
			<td>F1201</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>Olympus IX 71</td>
			<td></td>
		</tr>
		<tr>
			<td>Langendorff system</td>
			<td>Beijing Syutime Technology Co</td>
			<td>PlexiThermo-S-LANGC</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Marchauser</td>
			<td>MM33 links</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion chamber</td>
			<td>IonOptix</td>
			<td>FHD</td>
			<td></td>
		</tr>
		<tr>
			<td>Valve Controlled Gravity Perfusion System</td>
			<td>ALA</td>
			<td>VC 3-8</td>
			<td></td>
		</tr>
		<tr>
			<td>valve commander software</td>
			<td>ALA</td>
			<td>VC 3 1.0.1.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision flow regulator</td>
			<td>Delta Med</td>
			<td>3204315</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-Barrel Manifold Perfusion Pencil</td>
			<td>AutoMate Scientific</td>
			<td>04-08-[360]</td>
			<td></td>
		</tr>
		<tr>
			<td>Micron Removable Tip</td>
			<td>AutoMate Scientific</td>
			<td>360um i.d.</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence Measurement and Cell Dimensioning Systems</td>
			<td>IonOptix</td>
			<td>Hyperswitch</td>
			<td></td>
		</tr>
		<tr>
			<td>Recording software</td>
			<td>IonOptix</td>
			<td>IonWizard 6.2.59</td>
			<td></td>
		</tr>
		<tr>
			<td>Stimulator</td>
			<td>IonOptix</td>
			<td>MyoPacer EP</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague-dawley rats</td>
			<td>Beijing Vital River Laboratory Animal Technology Co.</td>
			<td>SCXK 2016-01-007436</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of sarcoplasmic reticulum calcium reserve and intracellular diastolic calcium removal in isolated ventricular cardiomyocytes

细胞内钙循环对心肌细胞收缩和舒张功能的调节起着至关重要的作用。心脏肌网 (SR) 担当一个 Ca2 +水库为收缩, reuptakes 细胞内 Ca2 +在放松期间。用于心跳收缩的 SR Ca2 +准备金是确定的, 而移除胞内 Ca2 +对心脏舒张功能至关重要。在一些病理生理学条件下, 如糖尿病和心力衰竭, 受损的钙清除和 SR 钙2 +存储在心肌细胞可能涉及心脏功能障碍的进展。在这里, 我们描述一个协议来评估 SRCa2 +保留和舒张期 Ca2 +删除。简单地说, 一个单一的心肌细胞被酶分离, 并通过钙显像系统记录 Fura-2 所示的胞内钙2 +荧光。为了使用咖啡因诱导总 SR Ca2 +释放, 我们预设了一个自动灌注开关程序通过相互关联的刺激系统和灌注系统。然后用单指数曲线拟合法分析钙瞬变和咖啡因诱导的钙离子脉冲的衰变时间常数。因此, 评价了 SR ca2 +atp 酶 (SERCA) 和 Na+-ca2 +交换器 (NCX) 对舒张钙的贡献。

在这里, 我们举例说明链脲佐菌素 (DM) 和年龄匹配的大 (SD) 大鼠的糖尿病大鼠。8周大的雄性 SD 大鼠 (200 ±20克) 接受了单腹腔注射脲佐菌素 (70 毫克/千克, ip) 为 DM 组或枸橼酸的控制组的缓冲。一周后, 有血糖和 #62 的老鼠, 16.7 摩尔/L 被认为是糖尿病。8周后, 左心室肌细胞酶分离。在钙重新引入后, 大约有 70-80% 的心肌细胞存活在生存状态。只有杆状细胞, 清晰的条纹, 和稳定?...

Watch this Scientific Journal Video about Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes at JoVE.com

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

细胞内钙循环对心肌细胞收缩和舒张功能的调节起着至关重要的作用。在这里, 我们描述了一个协议, 以评估肌网钙2 +储备和舒张钙功能的心肌细胞钙显像系统。

肌网钙储备与细胞内舒张钙在心室肌细胞中的分离评价

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

Research

JoVE Journal

Medicine

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

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

在分离的小鼠使用红桃心功能和能量学的评估 31 P核磁共振

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

科研

医学

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

隔离与人类心室肌细胞的功能研究从新鲜手术样品

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

冠脉流量和冠状动脉血流储备用小鼠压力超负荷基于模型的超声评估

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Chronic Thromboembolic Pulmonary Hypertension and Assessment of Right Ventricular Function in the Piglet

慢性血栓栓塞性肺动脉高压和右心室功能的仔猪评估

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. ...

Transthoracic Speckle Tracking Echocardiography for the Quantitative Assessment of Left Ventricular Myocardial Deformation

经胸斑点跟踪超声心动图左室心肌变形的定量评价

The value of conventional echocardiography is limited by differences in inter-individual image interpretation and therefore largely dependent on the ...

Treatment with Vancomycin Loaded Calcium Sulphate and Autogenous Bone in an Improved Rabbit Model of Bone Infection

万古霉素滴入硫酸钙和自体骨治疗兔骨感染的改进模型

Bone infection results from bacterial invasion, which is extremely difficult to treat in clinical, orthopedic, and traumatic surgery. The bone infection ...

Echocardiographic Measurement of Right Ventricular Diastolic Parameters in Mouse

小鼠右心室舒张参数的超声心电图测量

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV ...

Invasive Hemodynamic Assessment for the Right Ventricular System and Hypoxia-Induced Pulmonary Arterial Hypertension in Mice

小鼠右心室系统及缺氧诱发肺动脉高血压的侵入性血液动力学评估

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. ...

Generation of Ventricular-Like HiPSC-Derived Cardiomyocytes and High-Quality Cell Preparations for Calcium Handling Characterization

生成心室样HiPSC衍生的心肌细胞和用于钙处理特征的高质量细胞制剂

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) ...

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

使用皮肤心肌细胞进行心脏功能的体外评估

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