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.
Cardiac hypertrophy, a process initiated by mechanical alterations, is hypothesized to cause long-term molecular-level alteration in the sarcomere lattice, which is the main force-generating component in the heart muscle. This molecular-level alteration is beyond the resolving capacity of common light microscopy. Second harmonic generation (SHG) microscopy has unique capability for visualizing ordered molecular structures in biological tissues without labeling. Combined with polarization imaging technique, SHG microscopy is able to extract structural details of myosin at the molecular level so as to reveal molecular-level alterations that occur during hypertrophy. The myosin filaments are believed to possess C6 symmetry; thus, the nonlinear polarization response relationship between generated second harmonic light I^2ωand incident fundamental light I^ω is determined by nonlinear coefficients, χ_15, χ_31 and χ_33. χ_31/χ_15 is believed to be an indicator of the molecular symmetry of myosin filament, whileχ_33/χ_15represents the intramyosin orientation angle of the double helix. By changing the polarization of the incident light and evaluating the corresponding SHG signals, the molecular structure of the myosin, reflected by the χ coefficients, can be revealed. With this method, we studied the structural properties of heart tissues in different conditions, including those in normal, physiologically hypertrophic (heart tissue from postpartum female rats), and pathologically hypertrophic (heart tissue from transverse-aorta constricted rats) conditions. We found that ratios of χ_31/χ_15 showed no significant difference between heart tissues from different conditions; their values were all close to 1, which demonstrated that Kleinman symmetry held for all conditions. Ratios of χ_33/χ_15 from physiologically or pathologically hypertrophic heart tissues were raised and showed significant difference from those from normal heart tissues, which indicated that the intramyosin orientation angle of the double helix was altered when heart tissues hypertrophied. Polarization-resolved SHG microscopy permitted us to study heart tissues at the molecular level and may serve as a diagnostic tool for cardiac hypertrophy.
Second harmonic generation (SHG) microscopy is a new imaging technique used in sarcomeric-addition studies. However, during the early stage of cell culture in which sarcomeric additions occur, the neonatal cardiomyocytes that we have been working with are very sensitive to photodamage, the resulting high rate of cell death prevents systematic study of sarcomeric addition using a conventional SHG system. To address this challenge, we introduced use of the pulse-splitter system developed by Na Ji et al. in our two photon excitation fluorescence (TPEF) and SHG hybrid microscope. The system dramatically reduced photodamage to neonatal cardiomyocytes in early stages of culture, greatly increasing cell viability. Thus continuous imaging of live cardiomyocytes was achieved with a stronger laser and for a longer period than has been reported in the literature. The pulse splitter-based TPEF-SHG microscope constructed in this study was demonstrated to be an ideal imaging system for sarcomeric addition-related investigations of neonatal cardiomyocytes in early stages of culture.
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.
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