Effect of Substrate Stiffness on Redox State of Single Cardiomyocyte: A Scanning Electrochemical Microscopy Study

Abstract

Mechanical microenvironment plays a key role in the regulation of the phenotype and function of cardiac cells, which are strongly associated with the intracellular redox mechanism of cardiomyocytes. However, the relationship between the redox state of cardiomyocytes and their mechanical microenvironment remains elusive. In this work, we used polyacrylamide (PA) gels with varying stiffness (6.5−92.5 kPa) as the substrate to construct a mechanical microenvironment for cardiomyocytes. Then we employed scanning electrochemical microscopy (SECM) to in situ characterize the redox state of a single cardiomyocyte in terms of the apparent rate constant (k f ) of the regeneration rate of ferrocenecarboxylic by glutathione (GSH) released from cardiomyocyte, which is the most abundant reactant of intracellular reductive-oxidative metabolic cycles in cells and can represent the redox level of cardiomyocytes. The obtained SECM results show that the cardiomyocytes cultured on the stiffer substrates present lower k f values than those on the softer ones, that is, the more oxidative state of cardiomyocytes on the stiffer substrates compared to those on the softer ones. It proves the relationship between mechanical factors and the redox state of cardiomyocytes. This work can contribute to understanding the intracellular chemical process of cardiomyocytes during physiopathologic conditions. Besides, it also provides a new SECM method to in situ investigate the redox mechanism of cardiomyocytes at a single-cell level.

Investigating the Effect of Substrate Stiffness on the Redox State of Cardiac Fibroblasts Using Scanning Electrochemical Microscopy

“…Then, we used SECM to monitor the GSH efflux rate from cardiac fibroblasts on PA gels with tunable stiffness. In the SECM measurements, FcCOOH was selected as the redox mediator since it cannot penetrate across cell membrane and thus would not affect the cellular redox state of cell. , In principle, the oxidized ferrocene carboxylate ([FcCOOH] + ) generated by the SECM probe diffuses to the cardiac fibroblast surface and reacts with the GSH released from cardiac fibroblasts to regenerate FcCOOH, which diffuses back to the SECM probe surface, resulting in a detectable increment of the probe current (Figure A). Through fitting the SECM experimental approach curves with the theoretical model, the kinetic constant ( k ) of the redox reaction between GSH and [FcCOOH] + can be obtained.…”

“…In the probe approach curve experiments, the probe applied with a potential of 0.5 V (FcCOOH as the redox mediator) or −0.35 V (Ru­(NH 3 ) 6 Cl 3 as the redox mediator) ( vs Ag/AgCl RE) was controlled to approach to the cell surface with an approach speed of 0.5 μm s –1 . For SECM imaging experiments with the depth scan mode, the probe applied with a potential of 0.5 V ( vs Ag/AgCl RE) was controlled to linearly scan above the cardiac fibroblast surface, that is, from 2 to 20 μm away from “the highest point” of cell surface in the z -direction with a controlling 0.5 μm interval (Figure A). , The current distribution patterns were obtained by recording the probe oxidation currents, and the probe approach curves were extracted from the depth scan images by drawing vertical lines above the cell center.…”

Research advances of scanning electrochemical microscopy in biomedical field

“…[26] . 下面按照以上分类介绍几种SECM在细胞表征 图 1 SECM在单细胞、细胞球和微组织领域的应用研究示意图 [15,52,64,84] (网络版彩图) 图 2 用于细胞研究的SECM系统装置示意图 (网络版彩图) [21,28] 和外排或消耗的活性氧物 质(如H 2 O 2 )等 [15,20] (图3(a)), 并且可进一步通过对铂微 电极表面修饰(如用酶或纳米材料)拓宽铂微电极检测 物质的种类、提高其检测灵敏度. 例如, 通过在铂微电 极上修饰葡萄糖氧化酶或乳酸氧化酶可提高其对细胞 代谢活性(如葡萄糖摄取量)的检测特异性和抗干扰能 力 [29,30] , 在铂微电极上修饰纳米材料(如石墨烯、碳纳 米管)可进一步提高其对细胞代谢产物(如H 2 O 2 )的检 测灵敏度 [31,32] .…”

“…(a) 直径25 μm的铂圆盘微电极 [53] ; (b) 玻璃管微电极 [39] ; (c) 碳/玻璃微管双 通道电极 [40] ; (d) 金/玻璃微管微圆环双通道电极 [25] ; (e) 直径114 nm的铂纳米电极 [51] ; (f) 玻璃管纳米电极 [52] (网络版彩图) [58] ; (b) 可编程-SECM表 征细胞原理示意图和对PC12细胞高度、膜通透性和呼吸作用三个参数的连续表征图像 [59] ; (c) 应用纳米电极检测单突触神经 递质的释放过程示意图和结果图 [52] (网络版彩图) [21] ; (b) SECM系统与局部给药和 细胞培养舱结合检测心肌细胞的搏动情况和耗氧量 [22] ; 应用SECM (c) 研究不同硬度基质上心肌细胞的GSH外排通量 [64] , (d) 健康和病理性hmMSCs细胞内的氧化还原状态 [66,67] (网络版彩图) Figure 6 Application examples of SECM in studies of cardiomyocytes. (a) Application of SECM to characterize the pulsation and oxygen consumption of cardiomyocytes [21]; (b) Application of SECM combined with micropipette injection system and cell culture chamber to detect the pulsation and oxygen consumption of cardiomyocytes [22]; Applications of SECM to investigate (c) the efflux of GSH released from cardiomyocytes cultured on the substrates with different stiffness [64] [69] ; (b) SECM用于检测细 胞表面SEAP表达的原理示意图和微孔阵列中HeLa细胞的荧光和SEAP表达的SECM图像 [71] ; (c) SECM研究GSH外排的原理示 意图和HeLa细胞GSH外排的 SECM图像 [8] ; (d) SECM检测膜渗透性的原理示意图及不同浓度CdCl 2 加入后T24细胞的光学显 微镜照片和FcMeOH扩散的SECM图像 [76] ; (e) SECM检测T24细胞释放H 2 O 2 的原理示意图和不同时间T24细胞外H 2 O 2 电流的 SECM图像 [15] ; (f) 应用镀铂碳纳米电极作SECM探针, 检测乳腺癌细胞(MDA-MB-231、MDA-MB-468、MCF-10A)内ROS/ RNS(H 2 O 2 、ONOO − 、NO和NO 2 − )的伏安图 [16] (网络版彩图) . 之后, Bard课题组 细胞表面不同位置对FcCH 2 OH渗透系数的一致性 [75] .…”

“…Schematic diagram of applications of SECM in studies of single cell, cell spheroid and microtissue[15,52,64,84] (color online).…”