Abstract 19317: Real-Time Visualization of Endogenous Titin Dynamics in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes
Introduction: The giant myofilament protein titin is a critical determinant of myofibril elasticity and sarcomere structure in striated muscle. Although significant advances have been made in our understanding of the biophysical and mechanical properties of titin, the mechanisms governing sarcomeric titin turnover are poorly understood. Our goal is to establish a cardiomyocyte cell culture model that would enable visualization of sarcomeric titin turnover, in real-time.
Methods and Results: To directly visualize titin incorporation and turnover in cardiac sarcomeres, we used CRISPR/Cas9 genome editing in human induced pluripotent stem cells (hiPSC) to knock-in a photoconvertible fluorescent protein, mEos3.2, into the C-terminus of titin to produce hiPSC-derived cardiomyocytes (hiPSC-CM) that express endogenous sarcomeric titin-mEos3.2 fusion proteins. Cardiomyocyte induction of the titin-mEos3.2 hiPSC line demonstrated that the titin-mEos3.2 fusion protein is expressed in the sarcomeres in the expected striated pattern without affecting function. Fluorescence recovery after photobleaching (FRAP) revealed that the titin-mEos3.2 sarcomere exchange half-life is 1.2 hours with a mobile fraction of 68% (Table 1). Inhibition of protein synthesis with CHX increased titin exchange half-life to 2.1 hours but did not affect the mobile fraction. Proteasome Inhibition with CFZ had no affect on titin exchange half-life but resulted in a ~20% decrease in titin mobility. Contractile arrest with BDM had no affect on either the titin mobile fraction or titin exchange rate. Whole hiPSC-CM cell photoconversion revealed that titin is actively recycled among myofibrils.
Conclusion: Our results imply that titin is a dynamic protein within the complex macromolecular structure of the cardiac sarcomere that is continuously undergoing dynamic exchange between sarcomeres in the contracting cardiomyocyte.
Author Disclosures: A.G. Cadar: None. T.K. Feaster: None. K.R. Bersell: None. L. Wang: None. D.M. Roden: Research Grant; Modest; NIH. B.C. Knollmann: None. C.C. Hong: None. C.C. Lim: None.
- © 2016 by American Heart Association, Inc.