Disassembly of Myofibrils in Adult Cardiomyocytes during Dedifferentiation Honghai Liu 1 , Wan Qin 1 , Yonghong Shao 2 , Zhonghai Wang 1 , Huaxiao Yang 1 , Raymond B. Runyan 3 , Thomas K. Borg 4 , and Bruce Z. Gao 1 * 1 Department of Bioengineering and COMSET, Clemson University, Clemson, SC 29634, USA 2 Institute of Optoelectronics, Shenzhen University, Shenzhen, Guangdong, PR China 3 Department of Cellular and Molecular Medicine, University of Arizona, Tucson, AZ, 85724, USA 4 Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC, 29425, USA *Corresponding author’s email: zgao@clemson.edu ABSTRACT Using hybrid TPEF-SHG imaging and immunocytological techniques, we studied dedifferentiation of adult cardiomyocytes. First, the myofibrils shrank to shorten the sarcomere length. At the cell ends, the striated pattern of myosin filaments began to dissociate; at the center of the cell, the striated pattern of alpha-actinin first faded away and reappeared near the cell membrane during dedifferentiation. The results suggest that when freshly isolated adult cardiomyocytes are used to model cardiac muscle, the end-to-end connection may be important to maintain their striated myofibrillar structure and rod-shape morphology. 1. INTRODUCTION Cultured primary adult cardiomyocytes have been used to model the adult myocardium and are increasingly important in cardiology and cardiopathology research. However, freshly dissected adult cardiomyocytes undergo dedifferentiation during culturing in vitro: They lose their rod-shape morphology and striated myofibril structure. Because of the consequent changes in physiological functions[1-5], dissected adult cardiomyocytes are not an ideal in vitro model. To improve the model’s validity, the striated, rod-shape morphology of the adult cardiomyocytes must be maintained. Our imaging technique based on two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) has demonstrated the advantages of intrinsic high-resolution and high-contrast in biomedical research[6] and, specifically, in study of dynamic remodel of myofibrils[7]. The double wavelength and high excitation power requirement of TPEF and SHG can achieve a deeper penetration inside biological materials with higher 3D resolution than conventional single- photon excitation microscopy[6, 8]. In our previous research[9-14], dynamic sarcomeric additions in a living cardiomyocyte have been recorded for up to several minutes through SHG technique[15]. However, dedifferentiation of adult cardiomyocytes is a process that spans hours to days. We have developed a hybrid TPEF-SHG imaging system with an on-stage incubator that provides conditions that are close to the in vitro culture condition of isolated cardiomyocytes (e.g., 5% CO 2 , 37ºC, 95% humidity)[16]. Here, we report application of our onstage incubator-combined TPEF-SHG imaging system to record the dedifferentiation process of living adult cardiomyocytes. During dedifferentiation, first the myofibrils shrank, shortening the length of the sarcomeres, then the striated structure of myofibrils dissociated from the cell ends, and the dissociation then extended to the whole cell. The results suggest that in vivo, the striated patterns of the different sarcomeric components may not dissociate simultaneously through the entire cell during dedifferentiation. Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XI, edited by Daniel L. Farkas, Dan V. Nicolau, Robert C. Leif, Proc. of SPIE Vol. 8587, 85871W © 2013 SPIE · CCC code: 1605-7422/13/$18 · doi: 10.1117/12.2005156 Proc. of SPIE Vol. 8587 85871W-1 Downloaded From: http://spiedigitallibrary.org/ on 03/11/2013 Terms of Use: http://spiedl.org/terms