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Updated: Jul 11, 2025
Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo
Published on: October 2, 2021
Visualization of cardiac thick filament dynamics in ex vivo heart preparations
Colleen M Kelly1, Jody L Martin2, Molly Coseno3
1Molecular Physiology and Biophysics Department, University of Vermont, Larner College of Medicine, Burlington, VT 05405, United States of America.
Insights
Cardiac thick filaments are surprisingly dynamic, allowing rapid molecular exchange within sarcomeres. This continuous turnover, crucial for heart cell function, occurs much faster than previously thought.
Area of Science:
- Cardiovascular Biology
- Muscle Physiology
- Molecular Dynamics
Background:
- Cardiac muscle cells are terminally differentiated and require continuous function throughout life.
- Sarcomeric thick filaments, composed of myosin, are essential for cardiac contraction but have a short protein half-life (~10 days).
- The mechanism for stochastic protein replacement within thick filaments remains unknown.
Purpose of the Study:
- To investigate the dynamic nature of cardiac thick filaments in vivo.
- To test the hypothesis that thick filament structure is highly dynamic to facilitate molecular replacement.
Main Methods:
- Adeno-associated virus (AAV) mediated transduction to introduce GFP-labeled myosin regulatory light chain (RLC-GFP) into adult mouse hearts.
- Fluorescence recovery after photobleaching (FRAP) using multiphoton microscopy to quantify RLC-GFP mobility.
- Biochemical analysis of RLC-GFP binding to myosin heavy chain after cardiomyocyte lysis.
Main Results:
- RLC-GFP was correctly localized within thick filaments and did not affect heart size or actin sliding.
- RLC-GFP molecules exhibited high mobility within sarcomeres on a minute timescale.
- RLC-GFP remained bound to myosin heavy chain, which adopted a compact configuration upon dissociation from filaments.
Conclusions:
- Cardiac thick filaments are highly dynamic structures in the intact heart.
- Molecular exchange within thick filaments occurs approximately 1500 times faster than predicted by protein synthesis/degradation rates.
- This dynamic turnover is essential for maintaining cardiac function despite rapid protein replacement.
Rationale:
Cardiac muscle cells are terminally differentiated after birth and must beat continually throughout one's lifetime. This mechanical process is driven by the sliding of actin-based thin filaments along myosin-based thick filaments, organized within sarcomeres. Despite costly energetic demand, the half-life of the proteins that comprise the cardiac thick filaments is ∼10 days, with individual molecules being replaced stochastically, by unknown mechanisms.
Objectives:
To allow for the stochastic replacement of molecules, we hypothesized that the structure of thick filaments must be highly dynamic in vivo.
Methods And Results:
To test this hypothesis in adult mouse hearts, we replaced a fraction of the endogenous myosin regulatory light chain (RLC), a component of thick filaments, with GFP-labeled RLC by adeno-associated viral (AAV) transduction. The RLC-GFP was properly localized to the heads of the myosin molecules within thick filaments in ex vivo heart preparations and had no effect on heart size or actin filament siding in vitro. However, the localization of the RLC-GFP molecules was highly mobile, changing its position within the sarcomere on the minute timescale, when quantified by fluorescence recovery after photobleaching (FRAP) using multiphoton microscopy. Interestingly, RLC-GFP mobility was restricted to within the boundaries of single sarcomeres. When cardiomyocytes were lysed, the RLC-GFP remained strongly bound to myosin heavy chain, and the intact myosin molecules adopted a folded, compact configuration, when disassociated from the filaments at physiological ionic conditions.
Conclusions:
These data demonstrate that the structure of the thick filament is highly dynamic in the intact heart, with a rate of molecular exchange into and out of thick filaments that is ∼1500 times faster than that required for the replacement of molecules through protein synthesis or degradation.
