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Updated: Aug 14, 2025
Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo
Published on: October 2, 2021
Fluorescence lifetime-based assay reports structural changes in cardiac muscle mediated by effectors of contractile
Alexey V Dvornikov1, Thomas A Bunch1, Victoria C Lepak1
1Department of Cellular and Molecular Medicine, University of Arizona , Tucson, AZ, USA.
Abstract:
Cardiac muscle contraction is regulated by Ca2+-induced structural changes of the thin filaments to permit myosin cross-bridge cycling driven by ATP hydrolysis in the sarcomere. In congestive heart failure, contraction is weakened, and thus targeting the contractile proteins of the sarcomere is a promising approach to therapy. However, development of novel therapeutic interventions has been challenging due to a lack of precise discovery tools. We have developed a fluorescence lifetime-based assay using an existing site-directed probe, N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD) attached to human cardiac troponin C (cTnC) mutant cTnCT53C, exchanged into porcine cardiac myofibrils. We hypothesized that IANBD-cTnCT53C fluorescence lifetime measurements provide insight into the activation state of the thin filament. The sensitivity and precision of detecting structural changes in cTnC due to physiological and therapeutic modulators of thick and thin filament functions were determined. The effects of Ca2+ binding to cTnC and myosin binding to the thin filament were readily detected by this assay in mock high-throughput screen tests using a fluorescence lifetime plate reader. We then evaluated known effectors of altered cTnC-Ca2+ binding, W7 and pimobendan, and myosin-binding drugs, mavacamten and omecamtiv mecarbil, used to treat cardiac diseases. Screening assays were determined to be of high quality as indicated by the Z' factor. We conclude that cTnC lifetime-based probes allow for precise evaluation of the thin filament activation in functioning myofibrils that can be used in future high-throughput screens of small-molecule modulators of function of the thin and thick filaments.
Insights
A new fluorescence lifetime assay using cardiac troponin C (cTnC) probes precisely measures thin filament activation. This tool aids in developing new therapies for heart failure by screening drug candidates targeting cardiac contractile proteins.
Area of Science:
- Biophysics
- Cardiovascular Physiology
- Drug Discovery
Background:
- Cardiac muscle contraction relies on Ca2+-induced thin filament structural changes for myosin cross-bridge cycling.
- Congestive heart failure involves weakened contraction, making sarcomere contractile proteins a therapeutic target.
- Developing new therapies is hindered by a lack of precise discovery tools for cardiac contractile function.
Purpose of the Study:
- To develop and validate a fluorescence lifetime-based assay for precise evaluation of thin filament activation.
- To assess the assay's sensitivity and precision in detecting structural changes in cardiac troponin C (cTnC).
- To determine the assay's utility in high-throughput screening for cardiac disease therapeutics.
Main Methods:
- A site-directed probe, N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD), was attached to a human cTnC mutant (cTnCT53C).
- The labeled cTnC was exchanged into porcine cardiac myofibrils, and fluorescence lifetime measurements were used to monitor thin filament activation.
- The assay's performance was evaluated using Ca2+ binding, known modulators (W7, pimobendan), and myosin-binding drugs (mavacamten, omecamtiv mecarbil).
Main Results:
- The IANBD-cTnC fluorescence lifetime assay successfully detected Ca2+ binding and myosin binding to the thin filament.
- The assay demonstrated sensitivity to known modulators of cTnC-Ca2+ binding and myosin-binding drugs.
- High assay quality was confirmed by the Z' factor, indicating suitability for screening.
Conclusions:
- Fluorescence lifetime probes of cTnC provide precise insights into thin filament activation in functioning myofibrils.
- This assay represents a valuable tool for future high-throughput screening of small-molecule modulators of cardiac thin and thick filament function.
- The developed assay can accelerate the discovery of novel therapeutic interventions for cardiac diseases.
