啮齿动物心肌细胞心脏收缩功能障碍和Ca2+ 瞬变分析
Summary
提出了一组协议，描述了通过肌节长度检测以及钙（Ca 2+）瞬时测量在分离的大鼠肌细胞中测量收缩功能。这种方法在心力衰竭动物模型中的应用也包括在内。
Abstract
收缩功能障碍和Ca2+ 瞬变通常在细胞水平上进行分析，作为心脏诱导损伤和/或重塑综合评估的一部分。评估这些功能改变的一种方法是在原发性成人心肌细胞中使用卸载缩短和Ca2+ 瞬时分析。对于这种方法，通过胶原酶消化分离成体肌细胞，使其具有Ca 2+ 耐受性，然后粘附在层粘连蛋白包被的盖玻片上，然后在无血清培养基中进行电起搏。一般方案利用成年大鼠心肌细胞，但可以很容易地调整来自其他物种的原代肌细胞。受伤心脏肌细胞的功能改变可以与假心肌细胞和/或 体外 治疗进行比较。该方法包括肌细胞起搏所需的基本要素，以及细胞室和平台组件。这种方法的详细协议包括通过肌节长度检测测量空载缩短和使用比率指示剂Fura-2 AM测量的细胞Ca2+ 瞬变以及原始数据分析的步骤。
Introduction
心脏泵功能分析通常需要一系列方法来获得足够的洞察力，特别是对于心力衰竭（HF）的动物模型。超声心动图或血流动力学测量可深入了解体内心功能不全1，而体外方法通常用于确定功能障碍是否由肌丝和/或负责将激发或动作电位与收缩功能偶联的 Ca2+ 瞬变（例如，激发-收缩 [E-C] 耦合）引起的。体外方法还提供了在寻求昂贵和/或费力的体内治疗策略之前筛选对神经激素、载体诱导的遗传改变以及潜在治疗剂的功能反应的机会2。
有几种方法可用于研究体外收缩功能，包括完整小梁3 或透化肌细胞4 中的力测量，以及在存在和不存在 HF5，6 的情况下完整肌细胞中的卸载缩短和 Ca2+ 瞬变。这些方法中的每一种都侧重于心肌细胞收缩功能，它直接负责心脏泵功能2，7。然而，收缩和E-C偶联的分析最常通过测量分离的Ca 2+耐受成年肌细胞中的肌肉长度缩短和Ca2+瞬变来进行。实验室利用详细的已发布方案从大鼠心脏中分离出肌细胞进行此步骤8。
Ca2+ 瞬时和肌丝都有助于完整肌细胞的缩短和再延长，并可能导致收缩功能障碍2，7。因此，当体外功能分析需要含有Ca2+循环机制和肌丝的完整肌细胞时，建议使用这种方法。例如，完整的分离肌细胞对于通过基因转移修饰肌丝或Ca2+循环功能后研究收缩功能是理想的9。此外，在研究下游第二信使信号通路的影响和/或对治疗剂的反应时，建议使用完整的肌细胞方法来分析神经激素的功能影响2。单个肌细胞中负荷依赖性力的替代测量最常在低温（≤15°C）膜透化（或蒙皮）后进行，以去除Ca2+瞬时贡献并关注肌丝功能10。完整肌细胞中负载依赖性力加上Ca2+瞬变的测量很少见，这主要是由于该方法11的复杂性和技术挑战，特别是当需要更高的通量时，例如测量对神经激素信号的反应或作为治疗剂的筛选。心脏小梁的分析克服了这些技术挑战，但也可能受到非肌细胞、纤维化和/或细胞外基质重塑的影响2。上述每种方法都需要含有成人肌细胞的制剂，因为新生儿肌细胞和来源于诱导多能干细胞（iPSC）的肌细胞尚未表达成人肌丝蛋白的全部补体，并且通常缺乏成人杆状肌细胞中存在的肌丝组织水平2。迄今为止，iPSCs的证据表明，在培养物中，向成体亚型的完全过渡超过134天12。
鉴于本系列的重点是HF，这些协议包括区分衰竭与非衰竭完整肌细胞收缩功能的方法和分析。代表性的例子来自在肾上缩窄后18-20周研究的大鼠肌细胞，如前所述5，13。然后与假处理的大鼠的肌细胞进行比较。
此处描述的协议和成像平台用于分析和监测HF发展过程中杆状心肌细胞中缩短和Ca2+ 瞬变的变化。对于该分析，将2 x 104 Ca 2+耐受的杆状肌细胞接种在22mm 2 层粘连蛋白涂层玻璃盖玻片（CSs）上并培养过夜，如前所述8。材料 表中提供了为该成像平台组装的组件，以及用于最佳成像的培养基和缓冲液。此处还提供了使用软件进行数据分析的指南和代表性结果。整个方案分为单独的子部分，前三个部分侧重于分离的大鼠肌细胞和数据分析，然后是细胞Ca 2 + 瞬时实验和肌细胞数据分析。
Protocol
对啮齿动物进行的研究遵循公共卫生服务关于人道护理和使用实验动物的政策，并获得了密歇根大学机构动物护理和使用委员会的批准。在这项研究中，从体重≥200g5的3-34个月大的Sprague-Dawley和F344BN大鼠中分离出肌细胞。使用男性和女性比率。 1. 肌细胞起搏用于收缩功能研究 为每组分离的肌细胞制作新鲜的基于M199的培养基（材料?…
Representative Results
从分离后的第二天（第2天）开始至隔离后4天对大鼠肌细胞进行收缩功能研究。虽然可以在分离后的第二天（即第2天）记录肌细胞，但在基因转移或处理后通常需要更长的培养时间以改变收缩功能8。对于分离后培养超过18小时的肌细胞，第1节中描述的起搏方案有助于维持T小管和一致的缩短和重新延长结果。 图 1A显示了含有用于缩短研…
Discussion
步骤1中概述的慢性起搏方案延长了研究分离的肌细胞和评估长期治疗影响的有用时间。在我们的实验室中，当使用慢性起搏的肌细胞上的肌节长度测量收缩功能时，隔离后长达 4 天获得了一致的结果。然而，当使用超过 1 周的培养基来调整心肌细胞时，肌细胞收缩功能会迅速恶化。
对于收缩功能研究，数据在37°C下收集，这接近大鼠的体温。为了优化肌细胞活力并获得每个?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了美国国立卫生研究院（NIH）拨款R01 HL144777（MVW）的支持。
Materials
| MEDIA | |||
| Bovine serum albumin | Sigma (Roche) | 3117057001 | Final concentration = 0.2% (w/v) |
| Glutathione | Sigma | G-6529 | Final concentration = 10 mM |
| HEPES | Sigma | H-7006 | Final concentration = 15 mM |
| M199 | Sigma | M-2520 | 1 bottle makes 1 L; pH 7.45 |
| NaHCO3 | Sigma | S-8875 | Final concentration = 4 mM |
| Penicillin/streptomycin | Fisher | 15140122 | Final concentration = 100 U/mL penicillin, 100 μg/mL streptomycin |
| REAGENTS SPECIFICALLY FOR Ca2+ IMAGING | |||
| Dimethylsulfoxide (DMSO) | Sigma | D2650 | |
| Fura-2AM | Invitrogen (Molecular Probes) | F1221 | 50 μg/vial; Prepare stock solution of 1 mM Fura-2AM + 0.5 M probenicid in DMSO; Final Fura2-AM concentration in media is 5 μM |
| Probenicid | Invitrogen (Fisher) | P36400 | Add 7.2 mg probenicid (0.5 M) to 1 mM Fura-2AM stock; Final concentration in media is 2.5 mM |
| MATERIALS FOR RAT MYOCYTE PACING | |||
| #1 22 mm2 glass coverslips | Corning | 2845-22 | |
| 3 x 36 inch cables with banana jacks | Pomona Electronics | B-36-2 | Supplemental Figure 1, panel C |
| 37oC Incubator with 95% O2:5% CO2 | Forma | 3110 | Supplemental Figure 1, panel E. Multiple models are appropriate |
| Class II A/B3 Biosafety cabinet with UV lamp | Forma | 1286 | Multiple models are appropriate |
| Forceps – Dumont #5 5/45 | Fine Science Tools | 11251-35 | |
| Hot bead sterilizer | Fine Science Tools | 1800-45 | |
| Low magnification inverted microscope | Leica | DM-IL | Position this microscope adjacent to the incubator to monitor paced myocytes for contraction at the start of pacing and after media changes; 4X and 10X objectives recommended |
| Pacing chamber | Custom | Supplemental Figure 1, panel A. The Ionoptix C-pace system is a commercially available alternative or see 22 | |
| Stimulator | Ionoptix | Myopacer | Supplemental Figure 1, panel D. |
| MATERIALS FOR CONTRACTILE FUNCTION and/or Ca2+ IMAGING ANALYSIS | ID in Supplemental Figure 2 & Alternatives/Recommended Options | ||
| Additional components for Ca2+ imaging analysis | Ionoptix | Essential system components: — Photon counting system – Xenon power supply with dual excitation light source – Fluorescence interface | - The photon counting system contains a photomultiplier (PMT) tube and dichroic mirrorand is installed adjacent to the CCD camera (panel A #4). – The power supply for the xenon bulb light source (see panel A #5 and panel C, left) is integrated with a dual excitation interface (340/380 nm excitation and 510 nm emission) shown in panel A #6. – The fluorescence interface between the computer and light source is shown in panel B, #12. |
| CCD camera with image acquisition hardware and software (240 frames/s) | Ionoptix | Myocam with CCD controller | Myocam and CCD controller are shown in Supplemental Fig. 2, panel A #4 and panel A #5 & panel C #5 (right), respectively. The controller is integrated with a PC computer system (panel B #14). |
| Chamber stimulator | Ionoptix | Myopacer | Panel B, #13; Alternative: Grass model S48 |
| Coverslip mounted perfusion chamber | Custom chamber for 22 mm2 coverslip with silicone adapter and 2-4 Phillips pan-head #0 screws (arrow, panel F) | Panel A #10 & panel F; Chamber temperature is calibrated to 37oC using a TH-10Km probe and the TC2BIP temperature controller (see temperature controller). Commercial alternatives: Ionoptix FHD or C-stim cell chambers; Cell MicroControls culture stimulation system | |
| Dedicated computer & software for data collection and analysis of function/Ca2+ transients | Ionoptix | PC with Ionwizard PC board and software | Panel B, #14; Contractile function is measured using either SarcLen (sarcomere length) or SoftEdge (myocyte length) acquisition modules of the IonWizard software. The Ionwizard software also includes PMT acquisition software for ratiometric Ca2+ imaging in Fura-2AM-loaded myoyctes. – A 4 post electronic rack mount cabinet and shelves are recommended for housing the somputer and cell stimulator. The fluorescence interface for Ca2+ imaging also is housed in this cabinet (see below). |
| Forceps – Dumont #5 TI | Fine Science Tools | 11252-40 | Panel F |
| Insulated tube holder for media | Custom | Panel A #9; This holder is easily assembled using styrofoam & a pre-heated gel pack to keep media warm | |
| Inverted brightfield microscope | Nikon | TE-2000S | Install a rotating turret for epi-fluorescence (Panel A #2) for Ca2+ imaging. A deep red (590 nm) condenser filter also is recommended to minimize fluorescence bleaching during Ca2+ imaging. |
| Isolator Table | TMC Vibration Control | 30 x 36 inches | Panel A, #1; Desirable: elevated shelving, Faraday shielding |
| Microscope eyepieces & objective | Nikon | 10X CFI eyepieces 40X water CFI Plan Fluor objective | Panel A #3; 40X objective: n.a. 0.08; w.d. 2 mm. A Cell MicroControls HLS-1 objective heater is mounted around the objective (see temperature controller below). NOTE: water immersion dispensers also are now available for water-based objectives. |
| Peristaltic pump | Gilson | Minipuls 3 | Panel A #8 and panel E |
| small weigh boat | Fisher | 08-732-112 | |
| Temperature controller | Cell MicroControls | TC2BIP | Panel A #7; Panel D. This temperature controller heats the coverslip chamber to 37oC. A preheater and objective heater are recommended for this platform. A Cell MicroControls HPRE2 preheater and HLS-1 objective heater are controlled by the TC2BIP temperature controller for our studies. |
| Under cabinet LED light with motion sensor | Sylvania | #72423 LED light | Recommended for data collection during Ca2+ transient imaging under minimal room light.. Alternative: A clip on flashlight/book light with flexible neck – multiple suppliers are available. |
| Vacuum line with in-line Ehrlenmeyer flask & protective filter | Fisher | Tygon tubing – E363; polypropylene Ehrlenmeyer flask – 10-182-50B; Vacuum filter – 09-703-90 | Panel A #11 |
References
- Reihle, C., Bauersachs, J. Small animal models of heart failure. Cardiovascular Research. 115 (13), 1838-1849 (2019).
- Peter, A. K., Bjerke, M. A., Leinwand, L. A. Biology of the cardiac myocyte in heart disease. Molecular Biology of the Cell. 27 (14), 2149-2160 (2016).
- Rossman, E. I., et al. Abnormal frequency-dependent responses represent the pathophysiologic signature of contractile failure in human myocardium. Journal of Molecular and Cellular Cardiology. 36 (1), 33-42 (2004).
- McDonald, K. S., Hanft, L. M., Robinett, J. C., Guglin, M., Campbell, K. S. Regulation of myofilament contractile function in human donor and failing hearts. Frontiers in Physiology. 11, 468 (2020).
- Ravichandran, V. S., Patel, H. J., Pagani, F. D., Westfall, M. V. Cardiac contractile dysfunction and protein kinase C-mediated myofilament phosphorylation in disease and aging. The Journal of General Physiology. 151 (9), 1070-1080 (2019).
- Chaudhary, K. W., et al. Altered myocardial Ca2+ cycling after left ventricular assist device support in failing human heart. Journal of the American College of Cardiology. 44 (4), 837-845 (2004).
- Walker, C. A., Spinale, F. G. The structure and function of the cardiac myocyte: A review of fundamental concepts. The Journal of Thoracic and Cardiovascular Surgery. 118 (2), 375-382 (1999).
- Lang, S. E., Westfall, M. V. Gene transfer into cardiac myocytes. Methods in Molecular Biology. 1299, 177-190 (2015).
- Davis, J., et al. Designing heart performance by gene transfer. Physiological Reviews. 88 (4), 1567-1651 (2008).
- Sweitzer, N. K., Moss, R. L. Determinants of loaded shortening velocity in single cardiac myocytes permeabilized with a-hemolysin. Circulation Research. 73 (6), 1150-1162 (1993).
- Yasuda, S., et al. Unloaded shortening increases peak of Ca2+ transients but accelerates their decay in rat single cardiac myocytes. American Journal of Physiology-Heart and Circulatory Physiology. 285 (2), 470-475 (2003).
- Racca, A. W., et al. Contractile properties of developing human fetal cardiac muscle. The Journal of Physiology. 594 (2), 437-452 (2016).
- Kim, E. H., et al. Differential protein expression and basal lamina remodeling in human heart failure. Proteomics Clinical Applications. 10 (5), 585-596 (2016).
- Eckerle, L. G., Felix, S. B., Herda, L. R. Measurement of antibody effects on cellular function of isolated cardiomyocytes. Journal of Visualized Experiments. (73), e4237 (2013).
- Robbins, H., Koch, S. E., Tranter, M., Rubinstein, J. The history and future of probenecid. Cardiovascular Toxicology. 12 (1), 1-9 (2012).
- Sakthivel, S., et al. In vivo and in vitro analysis of cardiac troponin I phosphorylation. Journal of Biological Chemistry. 280 (1), 703-714 (2005).
- Qi, M., et al. Downregulation of sarcoplasmic reticulum Ca2+-ATPase during progression of left ventricular hypertrophy. The American Journal of Physiology. 272 (5), 2416-2424 (1997).
- Sonnenblick, E. H., Spiro, D., Spotnitz, H. M. The ultrastructure basis of Starling’s law of the heart: the role of the sarcomere is determining ventricular size and stroke volume. American Heart Journal. 68 (3), 336-346 (1964).
- Kabaeva, Z., Zhao, M., Michele, D. E. Blebbistatin extends culture life of adult mouse cardiac myocytes and allows efficient and stable transgene expression. American Journal of Physiology-Heart and Circulatory Physiology. 294 (4), 1667-1674 (2008).
- Zak, R. Metabolism of myofibrillar proteins in the normal and hypertrophic heart. Basic Research in Cardiology. 72 (2-3), 235-240 (1977).
- Palmer, B. M., Thayer, A. M., Snyder, S. M., Moore, R. L. Shortening and [Ca2+] dynamics of left ventricular myocytes isolated from exercise-trained rats. Journal of Applied Physiology. 85 (6), 2159-2168 (1998).
Cite This Article
Lavey, E. A., Westfall, M. V. Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes. J. Vis. Exp. (183), e64055, doi:10.3791/64055 (2022).