Name: assessment of sarcoplasmic reticulum calcium reserve and intracellular diastolic calcium removal in isolated ventricular cardiomyocytes
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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 、 9 、 10 、 11 . 
<ol> <li> 设置 Langendroff 灌注系统。用正常的 Tyrode (NT) 溶液填充灌注系统, 将温度设置在36.5 和 #176 ° C, 并消除管内的气泡. </li>
	<li> 用水合氯醛 (400 毫克/千克) 腹腔注射 (ip) 称量和麻醉大鼠。通过评估5分钟后的尾部或脚趾夹紧反应, 确定麻醉状态. </li>
	<li> 在剑过程中用外科剪刀打开腹腔, 提起剑过程, 用剪刀打开胸腔。取出心包, 用弯曲的镊子稍稍抬起心脏, 识别主动脉弓, 用剪刀从主动脉根部切断心脏. </li>
	<li> 将心脏移至 60 mm 的盘子中, 用 NT 溶液冲洗。用两个微解剖钳握住主动脉, 然后将其安装到共生灌注套管上, 然后用外科缝合线将主动脉牢牢地结扎在套管上. 
	注: 熟练操作者可在十五年代内完成此过程. </li>
	<li> 用30毫升 NT 溶液灌注心脏, 以恢复冠状动脉的循环。然后, 将液切换到30毫升 Ca 2 + -免费 Tyrode 解决方案 (10 mM 牛磺酸, 1 毫克/毫升 BSA) 来停止心跳。接下来, 将液转换为胶原酶, 用于酵素消化的隔离液 (0.6 毫克/毫升). 
	注: 胶原酶 A 用于糖尿病大鼠实验, 或胶原酶 II. 的实验, 无糖尿病大鼠. </li>
	<li> 消化20-25 分钟后, 迅速将灌注液转化为 Ca 2 + 无 Tyrode 溶液, 以停止进一步消化。然后, 用镊子握住心脏, 将其从套管中切下来, 将心脏放在包含 KB 解决方案的 35 mm 盘中 (请参见 表 1 ). </li>
	<li> 用剪刀和镊子解剖 LV 墙。切断心房、右心室和房室交界区。剩余的组织是 LV;把它转换成一个新的35毫米的菜与 KB 解决方案。将 LV 组织切成小块. </li>
	<li> 用过滤过的塑料滴管轻轻地吸管, 并在10毫升 KB 解决方案中 re-suspend. </li>
	<li> 过滤150和 #181 的单元格; m 口径不锈钢过滤器, 并转移到15毫升离心管。离心机在 150 x g 为三十年代和放弃上清液。在10毫升 kb 溶液中重新悬浮心肌细胞, 自由沉淀6分钟, 丢弃上清液, 在10毫升 kb 溶液中 Re-suspend 颗粒. 
	注: 所有步骤均在36和 #176; C 在解决方案中以 100% O 2 进行了毒气. </li>
</ol> 3。钙重新引入 <ol> <li> 在 KB 解决方案中解决20分钟后, 丢弃上清, 并使用钙重新引入解决方案 A (0.3 mM ca 2 + 、4.5 ml ca 2 + 无 Tyrode 解决方案、1.5 ml NT 解决方案) re-suspend 肌细胞为 10 min. </li>
	<li> 重复上述过程与钙重新引入解决方案 B (0.3 mM ca 2 + , 3 毫升 ca 2 + 免费 Tyrode 解决方案, 3 毫升 NT 解决方案). </li>
	<li> 使用 NT 解决方案 (1.2 mM Ca 2 + ) 重复上述步骤, 以净化可用的肌细胞。将隔离的 LV 肌细胞储存在这个溶液中, 并在4-6 小时内进行研究. </li>
</ol> 4。设置灌注系统 <ol> <li> 将流入管与 NT 解决方案连接到腔灌注 ( 图 1A )。
	<li> 用于靶肌细胞的针灌注, 连接多流形尖端 ( 如 , 灌注铅笔, 称为铅笔), 固定在微, 对阀控灌注系统。添加 NT 解决方案和10毫米咖啡因溶液到铅笔的每一列 ( 图 1A ). </li>
	<li> 疏散管内的气泡, 避免空气堵塞. </li>
	<li> 计算十年代铅笔微米尖端的滴水数, 并手动调整流量调节器以使流速保持在大约 3 drip/10 的速度. </li>
</ol> 5。细胞内钙的测量 2 + 瞬时和肌缩短 7 , 8 , 9 <ol> 吸管10和 #181; L 细胞悬浮在幻灯片上,通过显微镜下的细胞计数器计数细胞数, 计算密度。将心肌细胞稀释到5万细胞/毫升的近似浓度. </li>
	<li> 将 Fura-2 乙酰 (AM) 股票 (钙敏感染料) 添加到1毫升的肌细胞悬浮液中, 使最终浓度为2和 #181; m 在室温下保持20分钟. </li>
	<li> 离心机在150克, 三十年代, 并 re-suspend 的心肌细胞与 NT 解决方案2次. </li>
	<li> 关闭指示灯并将电池保持在黑暗中。将心肌细胞放置在灌注室15分钟。然后启动腔灌注 (1.5 毫升/分钟) 与 NT 解决方案。使用刺激器 (波持续时间4.0 毫秒, 脉冲振幅 8.0 V) 对1赫兹场刺激的肌细胞进行5分钟的速度. </li>
	<li> 选择一个具有良好形状 (杆形, 锐边, 清晰的交叉条纹) 和稳定的刺激抽搐 (没有自发收缩) 下的10x 显微镜物镜。然后, 将显微镜放大倍数改变为 40x, 旋转 CCD 摄像机方向, 使细胞保持水平位置. </li>
	<li> 通过调整单元格框架适配器来框架单个心肌细胞。确保背景清晰. </li>
	<li> 将心肌细胞暴露在氙气灯发出的光中, 用340或 380 nm 波长的励磁滤镜, 并通过40x 目标对心肌细胞进行成像。510 nm 检测荧光信号。同时, 注意肌细胞的肌长度变化, 同时使用柔模块记录. </li>
	<li> 用双激发荧光光电倍增系统记录荧光。运行用于钙成像系统的录音程序, 单击并 #34; 文件/新文件和 #34; 要创建新的录制文件, 然后单击和 #34; 启动和 #34; 按钮同步记录荧光和肌长度. </li>
</ol> 6。评估 SR ca 2 + 保留和舒张期 ca 2 + 删除功能 7 8 9 <ol> <li> 互为 #34; 辅助外 #34; 端口与 #34 的刺激器; 模拟在和 #34; 阀门控制系统上的端口 ( 例如 , 阀门指挥官, 通过 BNC 缆线查看 材料表 ) (以同步 TTL 信号). </li>
	<li> 预设用于自动切换灌注阀的程序, 如前所述 8 、 9 ;下面列出了操作的详细步骤。
	<ol> <li> 根据 表 2 预设阀门控制软件中的参数, 然后单击 #34;D ownload 和 #34; 按钮下载程序中加载的序列。单击 #34; 特里格er 和 #34; 按钮以启用触发器函数. 
		注: 阀门控制系统可在接收 TTL 信号后执行序列. </li>
		<li> 将刺激程序预设为序列模式, 并按 表3设置参数 . </li>
	</ol> </li> <li> 将刺激器设置为和 #34; S1 步骤和 #34; 以1赫兹的速度使 LV 肌细胞加速. 用 NT 溶液以1.5 毫升/分钟的速度启动针灌注. 
	注: 由于针流速度快于腔内灌注, 针流的光折射不同于腔内灌注的光折射。光折射的差异表明了有效的针灌注范围, 它包围了靶细胞. </li>
	<li> 在低功耗显微镜下选择目标心肌细胞 (在下游序列中), 并确保它可以通过铅笔的微尖端达到。将显微镜放大倍数更改为400x。旋转 CCD 相机的方向, 以保持在水平位置的心肌细胞。调整单元格框架适配器下的矩形光圈, 使其适合于填充心肌细胞的适当窗口。确保有最小的背景;不要让其他心肌细胞或死细胞碎片在这个窗口. </li>
	<li> 调整固定在微上的铅笔的位置, 并在400x 显微镜放大倍数下仔细地将微尖端放在视野半径距离内的目标肌细胞上. </li>
	<li> 调整针流范围以覆盖目标心肌细胞, 并确保心肌细胞不能被针流扫走. </li>
	<li> 六十年代基本起搏后, 将刺激光标滚动至 #34;D 2 步和 #34;. 
	注意: 协议的其余部分将由刺激器和阀门控制系统自动执行。基于上述设置, 协议可以自动执行, 如 图 2A , 1 Hz 基本起搏与 NT 解决方案的六十年代。然后暂停起搏和延迟2秒, 迅速切换到10毫米咖啡因灌注十五年代 (功能释放 Ca 2 + 存储在 SR), 然后切换回 NT 解决方案. </li>
	<li> 在录制结束时, 检测单个心肌细胞的背景荧光。单击 "#34";p "#34" 按钮以暂停文件录制, 将显微镜视图移到附近的空白区域。单击 #34; 记录和 #34; 按钮以恢复录制秒数, 并记录背景校正的数值340和 380 nm 强度值. </li>
	<li> 打开和 #34; 操作/常量和 #34; 对话框, 并将这些值分别填入和 #34; 背景和 #34; 表格; 该软件可以通过减去背景值来校正 Fura 2 的比值. </li>
</ol> 7。数据分析 9 <ol> <li> 在基本起搏阶段测量钙瞬变和肌缩短, 平均抽搐脉冲, 然后分析动态参数, 如钙瞬态, 时间常数钙瞬变衰变 (Tau-1 Hz), 自动使用软件. 
	注意: 如果软件不能很好地适应衰变段, 则导出手动单指数曲线拟合的衰落跟踪. </li>
	<li> 对于咖啡因诱导的钙脉冲, 只选择一个陡波的脉冲来分析钙的去除功能。排除信号干扰、异常脉冲或中途流出的细胞. </li>
	<li> 测量咖啡因引起的钙脉冲的振幅 (钙咖啡因, SRCa 的指数 2 + 保留). </li>
	<li> 通过单指数曲线拟合 (十年代持续时间) 从软件中手动获取咖啡因诱导的钙脉冲 (Tau 咖啡因) 的衰变时间常数 ( 图 2C ). </li>
</ol>

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作者没有什么可透露的。

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。在我们的实验中, 10 毫米咖啡因的快速应用旨在诱导总 ca2 +释放, 并通过打开 RyR 通道来防止 SR 中的 ca2再。因此, 咖啡因诱导的 ca2 +脉冲的振幅可以作为 SR ca2 +保留的索引。此外, 在1赫兹和3赫兹的基本起搏中, 我们可以进一步研究 SR 负载的频率依赖性及其底层机制6。
在细胞质中去除 Ca2 +对心脏舒张功能至关重要。如图 2A所示, 在 1 Hz 场刺激的阶段, 钙瞬变的下降归因于 SERCA 在 SR, NCX 在细胞膜, 和慢传输系统 (线粒体 Ca2 + uniporter 和心肌 Ca2 +atp 酶)。由于慢速机制的贡献微不足道, Ca2 +瞬态的衰减时间常数 (Tau-1 Hz) 反映了 SERCA 和 NCX7、8、9的组合活动。在咖啡因灌注阶段, SERCA 未能建立锶钙储备。因此, 咖啡因引起的钙脉冲 (Tau 咖啡因) 的下降主要归因于 NCX。相应地, NCX 作用是消极的与 Tau 咖啡因相关。NCX 的贡献舒张钙2 +清除是积极的, 与 Tau-1 赫兹/头咖啡因的比例。SERCA 的贡献是积极的与头咖啡因和 Tau-1 Hz 的差异7,8,9。
要成功完成该过程 (图 2), 应注意一些技术要点。首先, 建立一个自动化的系统是关键的是切换起搏和灌注系统的精确。预置程序能准确控制延时时间, 快速应用咖啡因灌注。其次, 在这个过程中, 心肌细胞有被灌注液冲走的危险。为保持心室肌细胞稳定地附着在腔盘上, 在记录前应放置在盘中超过15分钟。
此外, 建立一个稳定的针灌系统应用咖啡因是另一个关键的步骤。这一步骤有三关键点: (1) 快速通道开关和平滑针灌注, (2) 井控灌注流速, (3) 针尖与靶肌细胞之间的适当距离。不适当设置的流速, 尖端距离, 或干扰的解决方案通道开关可能导致无法获得一个可接受的咖啡因诱导钙脉冲。对于咖啡因诱导的钙离子脉冲, 只有具有陡波峰的脉冲才能被选择来分析钙的去除功能。
基于膜片钳系统的替代方法可以提供相对准确的数据。然而, 一些因素, 如内部整流 K+电流, 可能会影响准确性。此外, 先进的设备, 熟练的技师, 和时间消耗的更高的要求也可能限制膜片钳的应用。在某种程度上, 我们的协议有限制它不能提供直接的价值的 SR 负荷或活动的 SERCA 和 NCX。参数 (例如, 钙咖啡因, Tau 咖啡因) 只反映改变 SR 内容和贡献的舒张钙2 +删除间接。但是, 该协议具有方便、技术和设备限制少的优点。

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>012314</td><td></td></tr><tr><td>MgCl2</td><td>Alfa Aesar</td><td>012315</td><td></td></tr><tr><td>KCl</td><td>Alfa Aesar</td><td>11595</td><td></td></tr><tr><td>HEPEs</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>D-Glucose</td><td>Alfa Aesar</td><td>A16828</td><td></td></tr><tr><td>NaOH</td><td>Alfa Aesar</td><td>A18395</td><td></td></tr><tr><td>KOH</td><td>Alfa Aesar</td><td>A18854</td><td></td></tr><tr><td>KH2PO4</td><td>Alfa Aesar</td><td>011594</td><td></td></tr><tr><td>MgSO4</td><td>Alfa Aesar</td><td>3337</td><td></td></tr><tr><td>L-Glutamic</td><td>Alfa Aesar</td><td>A15031</td><td></td></tr><tr><td>Taurine</td><td>Alfa Aesar</td><td>A12403</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>E3889</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>LS004177</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, 和稳定的收缩选择记录 (图 1B)。如图 1A所示, 我们为心肌细胞设置了腔灌注和针灌注。灌注阀和起搏系统由预设程序自动切换, 如图 2A所述。细胞内钙荧光记录的钙显像系统。值被报告为平均± SEM。
与 SD 组相比, DM 组显示咖啡因诱导的钙脉冲 (钙咖啡因) (0.332 ±0.008 与. 0.276 ± 0.008, t 检验: p和 #60; 0.05) 和较低的分数钙释放 SR (Ca-1 Hz/钙咖啡因) (78.5 ± 1.5% vs 72.1 ± 1.0%, t 检验: p和 #60; 0.05) (图 2B)。从单指数曲线拟合得到 Tau-1 赫兹和 Tau 咖啡因;DM 组显示了咖啡因诱导的钙脉冲 (Tau 咖啡因) (1.822 ± 0.07 ms vs) 的较长的衰变时间常数. 2.896 ± 0.088 ms, t 测试, p & #60; 0.05), 在 Tau-1 Hz/头-咖啡因比率 (0.076 ± 0.003 vs) 中的较高级别.0.086 ± 0.003, t 测试, p和 #60; 0.05) 比 SD 组 (图 2D)。这些结果表明, 抑郁的 SR ca2 +保留和受损的 ca2 +清除糖尿病心肌细胞。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55797/55797fig1.jpg"> 
图1。灌注系统和孤立性左心室肌细胞的图示.(A) 室灌注和铅笔灌注的图示。箭头指示在灌注室中 NT 溶液的流向。铅笔提供的灌注周围的目标细胞与 NT 或咖啡因溶液。微米尖端可以自由操控在会议厅上方。(B) 具有杆状和透明横纹的孤立性 LV 肌细胞的显微观察。<a href="//ecsource.jove.com/files/ftp_upload/55797/55797fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55797/55797fig2.jpg"> 
图2。锶钙储备和钙清除功能的评价.(A) 自动切换灌注阀和起搏系统的协议说明。(B) 统计值 sr ca2 +保留和 sr 分数 ca2 +释放 1 Hz 刺激抽搐在 DM 和 SD 组。DM 组显示的 sr ca2 +准备金和 sr 分数 ca2 +版本显著减少, 而不是 SD 组。(C) 软件面板显示了一个手动的单指数曲线拟合, 用于咖啡因诱导的钙脉冲 (Tau 咖啡因) 的衰变时间常数。(D) 条形图比较 DM 和 SD 组之间的 tau 咖啡因和 Tau-1 Hz/tau 咖啡因。DM 组显示, tau 咖啡因和 Tau-1 比 SD 组有显著增加的价值。值 = 平均± SEM, t 测试执行, *p和 #60; 0.05 与 SD 组相比。<a href="//ecsource.jove.com/files/ftp_upload/55797/55797fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>解决</td>
			<td>内容 (mM)</td>
		</tr>
		<tr>
			<td>普通 Tyrode (NT) 解决方案</td>
			<td>135氯化钠, 5.4 氯化钾, 1.2 氯化镁2, 10 葡萄糖, 10 HEPES, 1.2 Na2HPO4, 1.2 CaCl2,将 pH 值与氢氧化钠调整为7.35</td>
		</tr>
		<tr>
			<td>Ca2 +免费 Tyrode 解决方案</td>
			<td>135氯化钠, 5.4 氯化钾, 1.2 氯化镁2, 10 葡萄糖, 10 HEPES, 1.2 Na2HPO4, 10 牛磺酸, 用氢氧化钠调整 pH 值至7.35</td>
		</tr>
		<tr>
			<td>胶原酶隔离液</td>
			<td>胶原酶 A 或胶原酶 II + Ca2 +游离 Tyrode 溶液</td>
		</tr>
		<tr>
			<td>KB 解决方案</td>
			<td>120 koh, 120 l-谷氨酸, 5 MgCl2, 10 HEPEs, 1 EGTA, 10 葡萄糖, 20 牛磺酸, 调整 pH 值到7.35 与 koh</td>
		</tr>
		<tr>
			<td>咖啡因灌注液</td>
			<td>10咖啡因在 NT 解决方案</td>
		</tr>
	</tbody>
</table>
表1。心室肌细胞分离和细胞灌注的解决方案。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>阀</td>
			<td>时间 (秒)</td>
		</tr>
		<tr>
			<td>2</td>
			<td>15</td>
		</tr>
		<tr>
			<td>1</td>
			<td>60</td>
		</tr>
	</tbody>
</table>
表2。阀控系统 (阀门控制器) 中的参数预设。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>面板显示</td>
			<td>评论</td>
		</tr>
		<tr>
			<td>4.0 毫秒持续时间</td>
			<td>电刺激场时间</td>
		</tr>
		<tr>
			<td>8.0 V</td>
			<td>电气电压</td>
		</tr>
		<tr>
			<td>S1 1.0 HZ 999s</td>
			<td>在起搏状态下找到目标心肌细胞</td>
		</tr>
		<tr>
			<td>D2 001s</td>
			<td>选择此步骤暂停起搏和延迟1s 后, 基底抽搐记录</td>
		</tr>
		<tr>
			<td>D3 015s *</td>
			<td>"*" 表示该刺激器可以输出 5V TTL 信号</td>
		</tr>
		<tr>
			<td>D4 060s</td>
			<td>完成咖啡因灌注和细胞恢复正常状态</td>
		</tr>
		<tr>
			<td>结束</td>
			<td>回滚到 S1 步骤</td>
		</tr>
	</tbody>
</table>
表3。刺激器中的参数预设。

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 ...

assessment-sarcoplasmic-reticulum-calcium-reserve-intracellular

Nanyang Technological University

Research

JoVE Journal

Medicine

10.1K 次观看.  Zhejiang University School of Medicine. 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。

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

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

The study of electrophysiological properties of cardiac ion channels with the patch-clamp technique and the exploration of cardiac cellular Ca2+ handling abnormalities requires isolated cardiomyocytes. In addition, the possibility to investigate myocytes from patients using these techniques is an invaluable requirement to elucidate the molecular basis of cardiac diseases such as atrial fibrillation (AF).1 Here we describe a method for isolation of human atrial myocytes which are suitable for both patch-clamp studies and simultaneous measurements of intracellular Ca2+ concentrations. First, right atrial appendages obtained from patients undergoing open heart surgery are chopped into small tissue chunks ("chunk method") and washed in Ca2+-free solution. Then the tissue chunks are digested in collagenase and protease containing solutions with 20 &mu;M Ca2+. Thereafter, the isolated myocytes are harvested by filtration and centrifugation of the tissue suspension. Finally, the Ca2+ concentration in the cell storage solution is adjusted stepwise to 0.2 mM. We briefly discuss the meaning of Ca2+ and Ca2+ buffering during the isolation process and also provide representative recordings of action potentials and membrane currents, both together with simultaneous Ca2+ transient measurements, performed in these isolated myocytes.

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

We describe the isolation of human atrial myocytes which can be used for intracellular Ca2+ measurements in combination with electrophysiological patch-clamp studies.

CA同时测量人的心房肌细胞分离<sup&gt; +</sup&gt;瞬变和膜电流

The study of  electrophysiological properties of cardiac ion channels with the patch-clamp  technique and the exploration of cardiac cellular Ca2+ ...

科研

生物学

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.

钙火花的评估在正常肌肉纤维

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

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.

鼠心肌细胞的分离及生理分析

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

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.

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 ...

医学

实时分析离体淋巴管中的钙和收缩力

Real-time Evaluation of Absolute, Cytosolic, Free Ca2+ and Corresponding Contractility in Isolated, Pressurized Lymph Vessels

The lymphatic vasculature, now often referred to as &#34;the third circulation,&#34; is located in many vital organ systems. A principal mechanical function of the lymphatic vasculature is to return fluid from extracellular spaces back to the central venous ducts. Lymph transport is mediated by spontaneous rhythmic contractions of lymph vessels (LVs). LV contractions are largely regulated by the cyclic rise and fall of cytosolic, free calcium ([Ca2+]i).
This paper presents a method to concurrently calculate changes in absolute concentrations of [Ca2+]i and vessel contractility/rhythmicity in real time in isolated, pressurized LVs. Using isolated rat mesenteric LVs, we studied changes in [Ca2+]i and contractility/rhythmicity in response to drug addition. Isolated LVs were loaded with the ratiometric Ca2+-sensing indicator Fura-2AM, and video microscopy coupled with edge-detection software was used to capture [Ca2+]i&#160;and diameter measurements continuously in real time.
The Fura-2AM signal from each LV was calibrated to the minimum and maximum signal for each vessel and used to calculate absolute [Ca2+]i. Diameter measurements were used to calculate contractile parameters (amplitude, end diastolic diameter, end systolic diameter, calculated flow) and rhythmicity (frequency, contraction time, relaxation time) and correlated with absolute [Ca2+]i measurements.

作者聚焦：淋巴水肿和高血压研究的创新方法

This protocol describes a method to simultaneously measure cytosolic, free calcium [Ca2+]i and vessel diameter in contracting lymph vessels in real time and then calculate absolute Ca2+ concentrations as well as contractility/rhythmicity parameters. This protocol can be used to study Ca2+ and contractile dynamics across a variety of experimental conditions.

实时评估隔离、加压淋巴管中的绝对、胞质、游离 Ca2+ 和相应的收缩力

The lymphatic vasculature, now often referred to as &#34;the third circulation,&#34; is located in many vital organ systems. A principal mechanical ...

免疫与感染

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.

的内肌浆网钙光学标测<sup&gt; 2+</sup&gt;和的Langendorff灌注兔心脏跨膜电位

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a ...

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca2+ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca2+ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca2+-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca2+ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca2+ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca2+. This tool is useful for investigation into the nuanced changes in SR Ca2+ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca2+ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca2+ indicator, D1SR, is used to detect Ca2+ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca2+ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca2+ movement.

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

基于FRET钙内波动培养平滑肌细胞使用的肌质网的测量共焦成像

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...

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

Heart failure and cardiac arrhythmias are the leading causes of mortality and morbidity worldwide. However, the mechanism of pathogenesis and myocardial malfunction in the diseased heart remains to be fully clarified. Recent compelling evidence demonstrates that changes in the myofilament Ca2+ sensitivity affect intracellular Ca2+ homeostasis and ion channel activities in cardiac myocytes, the essential mechanisms responsible for the cardiac action potential and contraction in healthy and diseased hearts. Indeed, activities of ion channels and transporters underlying cardiac action potentials (e.g., Na+, Ca2+ and K+ channels and the Na+-Ca2+ exchanger) and intracellular Ca2+ handling proteins (e.g., ryanodine receptors and Ca2+-ATPase in sarcoplasmic reticulum (SERCA2a) or phospholamban and its phosphorylation) are conventionally measured to evaluate the fundamental mechanisms of cardiac excitation-contraction (E-C) coupling. Both electrical activities in the membrane and intracellular Ca2+ changes are the trigger signals of E-C coupling, whereas myofilament is the functional unit of contraction and relaxation, and myofilament Ca2+ sensitivity is imperative in the implementation of myofibril performance. Nevertheless, few studies incorporate myofilament Ca2+ sensitivity into the functional analysis of the myocardium unless it is the focus of the study. Here, we describe a protocol that measures sarcomere shortening/re-lengthening and the intracellular Ca2+ level using Fura-2 AM (ratiometric detection) and evaluate the changes of myofilament Ca2+ sensitivity in cardiac myocytes from rat hearts. The main aim is to emphasize that myofilament Ca2+ sensitivity should be taken into consideration in E-C coupling for mechanistic analysis. Comprehensive investigation of ion channels, ion transporters, intracellular Ca2+ handling, and myofilament Ca2+ sensitivity that underlie myocyte contractility in healthy and diseased hearts will provide valuable information for designing more effective strategies of translational and therapeutic value.

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.

肌丝的Ca评估<sup&gt; 2+</sup&gt;灵敏度潜在心脏兴奋收缩耦联

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

肌网钙储备与细胞内舒张钙在心室肌细胞中的分离评价

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CA同时测量人的心房肌细胞分离

钙火花的评估在正常肌肉纤维

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使用皮肤心肌细胞进行心脏功能的体外评估

实时评估隔离、加压淋巴管中的绝对、胞质、游离 Ca²⁺ 和相应的收缩力

实时评估隔离、加压淋巴管中的绝对、胞质、游离 Ca²⁺ 和相应的收缩力

实时评估隔离、加压淋巴管中的绝对、胞质、游离 Ca²⁺ 和相应的收缩力

的内肌浆网钙光学标测

基于FRET钙内波动培养平滑肌细胞使用的肌质网的测量共焦成像

肌丝的Ca评估