Study of the sarcomeric addition process in a tissue-like cell construct under mechanical overload via TPEF-SHG imaging system

Ailin Wei; Zhonghai Wang; Zongming Yang; Shenghao Tan; Tong Ye; Yonghong Shao; Thomas K. Borg; Bruce Z. Gao

doi:10.1117/12.2546280

14 February 2020 Study of the sarcomeric addition process in a tissue-like cell construct under mechanical overload via TPEF-SHG imaging system

Ailin Wei, Zhonghai Wang, Zongming Yang, Shenghao Tan, Tong Ye, Yonghong Shao, Thomas K. Borg, Bruce Z. Gao

Author Affiliations +

Proceedings Volume 11244, Multiphoton Microscopy in the Biomedical Sciences XX; 112442E (2020) https://doi.org/10.1117/12.2546280
Event: SPIE BiOS, 2020, San Francisco, California, United States

Abstract

Using our lab built two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) hybrid confocal imaging system, we observed, for the first time, the dynamic sarcomeric addition process in a rat cardiomyocyte cell culture system; this culture system expressed in vivo-like myofibril structure and mimicked mechanical overload experienced in a heart muscle tissue. Micro-grooved topographic patterned substrates com- bined with electrical stimulation are used to achieve the in vivo-like myofibril structure. After cardiomyocytes aligned, longitudinal and transverse mechanical stretch was applied to cardiomyocytes in parallel or perpendicu- lar, respectively, to the direction of alignment via stretching the substrates to mimic mechanical overload. Z discs, in which alpha-actinin expressed, have been proposed to involve in the process. TPEF detected alpha-actinin that labeled with enhanced yellow florescent protein via plasmid transfection. SHG is intrinsic to noncentrosym- metric structures, thus was used to detect myosin, a polar molecule expressed in myofibril. Pulse splitter system and synchronized recording system was introduced on TPEF-SHG imaging system to reduce the photodamage during live cell imaging. In our study, TPEF-SHG imaging system was used to study the dynamic process of sarcomeric addition in in vivo-like culture model under mechanical overload. This microscopic technique is ideal for tracking sarcomeric components to successively assemble onto pre-exist myofibrils and for revealing the role of Z discs played in sarcomeric addition. Transition of Z discs from continuous to broadened striation and from broadened to uniform striation under stretch has been observed. We concluded that continuous Z discs is the place of new sarcomeric addition.

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Ailin Wei, Zhonghai Wang, Zongming Yang, Shenghao Tan, Tong Ye, Yonghong Shao, Thomas K. Borg, and Bruce Z. Gao "Study of the sarcomeric addition process in a tissue-like cell construct under mechanical overload via TPEF-SHG imaging system", Proc. SPIE 11244, Multiphoton Microscopy in the Biomedical Sciences XX, 112442E (14 February 2020); https://doi.org/10.1117/12.2546280

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KEYWORDS

Imaging systems

Beam splitters

Heart

Second-harmonic generation

Tissues

Microscopes

Two photon excitation microscopy

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