Sarcomere M-line visualized via Titin

Movie. Z-stack of live hiPSC-derived cardiomyocytes expressing mEGFP-tagged titin protein. Twelve days after the onset of differentiation, cells were plated on PEI and laminin coated glass and imaged in 3D on a spinning disk confocal microscope 23 days later (35 days total after the onset of differentiation). Cells were treated with 15 mM of the myosin inhibitor 2,3-Butanedione monoxime (BDM) to prevent beating during image acquisition. Movie starts at the bottom of the cells and ends at the top. Inset shows detail of titan in myofibrils. Scale bars, 10 µm.

Movie. ​Time-lapse movie of live hiPSC-derived cardiomyocytes expressing mEGFP-tagged titin protein. Twelve days after the onset of differentiation, cells were plated on PEI and laminin coated glass and imaged on a spinning disk confocal microscope 23 days later (35 days total after the onset of differentiation). A single plane of cells was imaged continuously with a 100 ms exposure time. Inset shows detail of titan in myofibrils. Scale bars, 10 µm. Movie plays in real time.

Observations

• Titin is the largest known protein. It stretches across the sarcomere from the Z-disk (at its N-terminus) to the M-line (at its C-terminus). Titin functions like a spring when under tension with the contraction of sarcomeres. Titin also plays a structural role in regulating the length of the thick filament within sarcomeres.
• In hiPSC-derived cardiomyocytes, titin tagged at its C-terminus with mEGFP localizes to thin lines that striate myofilaments, consistent with localization to the M-line of sarcomeres.
• In hiPSC-derived cardiomyocytes from the mEGFP tagged Titin line, the fluorescent signal localizes to thin lines that striate myofilaments. While Titin spans the entire length from the Z-disk to the M-line, the much smaller mEGFP only highlights the location of the tagged Titin C-terminus. During cardiomyocyte beating, the contraction of sarcomeres can be seen in the changes in spacing between striations, and some myofibrils buckle.