Multimodal SHG-2PF Imaging of Microdomain Ca2+-Contraction Coupling in Live Cardiac Myocytes
Multimodal SHG-2PF Imaging of Microdomain Ca2+-Contraction Coupling in Live Cardiac Myocytes
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DOI：
10.1161/circresaha.115.307919
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发表时间：
2016-01-22
影响因子：
20.1
通讯作者：
Chan, James W.
中科院分区：
文献类型：
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作者：
Awasthi, Samir;Izu, Leighton T.;Chan, James W.
基本原理：心肌细胞收缩是由Ca 2+与肌钙蛋白C结合引起的，这触发了跨桥动力冲程和肌节中的肌丝滑动。同步的Ca 2+释放导致整个细胞收缩，并且用当前的显微镜技术很容易观察到。然而，目前尚不清楚局部的Ca 2+释放，如Ca 2+火花和波，可以引起局部肌节收缩。现代成像方法缺乏测量活心肌细胞中微区Ca 2 +-收缩偶联的能力。目的：开发一种用于成像健康和疾病模型心肌细胞中肌节水平Ca 2 +-收缩偶联的方法。方法和结果：新鲜分离的心肌细胞负载Ca 2 +-指示剂fluo-4。使用配备有飞秒脉冲近红外激光的共聚焦显微镜，同时激发肌原纤维A带的二次谐波产生和fluo-4的双光子荧光。Ca 2+信号和肌节应变在空间和时间上具有短延迟。此外，Ca 2+火花和波引起收缩的亚细胞微域，揭示了以前低估的作用，这些事件中产生亚细胞应变在pneumocole。Ca 2+活性和肌节应变也在机械负荷下起搏心肌细胞成像，揭示自发的Ca 2+波和相关的局部收缩压力超负荷诱导cardiomy.Conclusions：多模态二次谐波产生2-光子荧光显微镜可以同时观察到的Ca 2+释放和机械应变在subsarcomere水平在活心肌细胞。该方法受益于无标记的性质，二次谐波产生，这使得A-波段成像独立的T-小管形态，同时与钙离子指示剂。二次谐波产生双光子荧光成像技术广泛应用于健康和疾病中钙离子收缩耦合和机械化学转导的研究。
Rationale: Cardiac myocyte contraction is caused by Ca2+ binding to troponin C, which triggers the cross-bridge power stroke and myofilament sliding in sarcomeres. Synchronized Ca2+ release causes whole cell contraction and is readily observable with current microscopy techniques. However, it is unknown whether localized Ca2+ release, such as Ca2+ sparks and waves, can cause local sarcomere contraction. Contemporary imaging methods fall short of measuring microdomain Ca2+-contraction coupling in live cardiac myocytes.Objective: To develop a method for imaging sarcomere level Ca2+-contraction coupling in healthy and disease model cardiac myocytes.Methods and Results: Freshly isolated cardiac myocytes were loaded with the Ca2+-indicator fluo-4. A confocal microscope equipped with a femtosecond-pulsed near-infrared laser was used to simultaneously excite second harmonic generation from A-bands of myofibrils and 2-photon fluorescence from fluo-4. Ca2+ signals and sarcomere strain correlated in space and time with short delays. Furthermore, Ca2+ sparks and waves caused contractions in subcellular microdomains, revealing a previously underappreciated role for these events in generating subcellular strain during diastole. Ca2+ activity and sarcomere strain were also imaged in paced cardiac myocytes under mechanical load, revealing spontaneous Ca2+ waves and correlated local contraction in pressure-overload-induced cardiomyopathy.Conclusions: Multimodal second harmonic generation 2-photon fluorescence microscopy enables the simultaneous observation of Ca2+ release and mechanical strain at the subsarcomere level in living cardiac myocytes. The method benefits from the label-free nature of second harmonic generation, which allows A-bands to be imaged independently of T-tubule morphology and simultaneously with Ca2+ indicators. Second harmonic generation 2-photon fluorescence imaging is widely applicable to the study of Ca2+-contraction coupling and mechanochemotransduction in both health and disease.