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Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart
Published on: September 13, 2011
Imaging Ca2+ nanosparks in heart with a new targeted biosensor
Wei Shang1, Fujian Lu, Tao Sun
1From the State Key Laboratory of Biomembrane and Membrane Biotechnology, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, Peking-Tsinghua Center for Life Sciences (W.S., F.L., T.S., J.X., Y.W., G.W., L.C., X.W., H.C.) and State Key Laboratory of Biomembrane and Membrane Biotechnology, College of Life Science (L.-L.L., S.-Q.W.), Peking University, Beijing, China; and Department of Physiology and Pharmacology, University of Bristol, City of Bristol, United Kingdom (M.B.C.).
Researchers developed a targeted biosensor to visualize tiny Ca2+ signals within cardiac cells. This new tool improves understanding of excitation-contraction coupling and heart disease mechanisms.
Area of Science:
- Cardiovascular Biology
- Molecular Cardiology
- Calcium Signaling
Background:
- Cardiac function relies on precise calcium (Ca2+) signaling within dyads, the specialized junctions between the sarcoplasmic reticulum and transverse tubules.
- Existing Ca2+ probes lack the specificity to measure these localized signals due to diffusion, limiting understanding of excitation-contraction coupling.
Purpose of the Study:
- To develop and utilize a novel, junction-targeted Ca2+ biosensor to investigate Ca2+ dynamics in the nanoscopic dyadic space.
- To gain new insights into the function of ryanodine receptors (RyRs) in cardiac excitation-contraction coupling.
Main Methods:
- Genetically engineered a Ca2+ biosensor (GCaMP6f) by fusing it to junctional proteins (triadin 1 or junctin) for targeted localization.
- Utilized adenovirus-mediated gene transfer to express the biosensor in cardiac cells, confirmed colocalization with t-tubules and RyRs.
- Employed confocal imaging to visualize and quantify Ca2+ transients at the dyadic level.
Main Results:
- The targeted biosensor (GCaMP6f-triadin 1/junctin) exhibited faster kinetics and enabled visualization of Ca2+ nanosparks, approximately 50 times smaller than conventional Ca2+ sparks.
- Measurements of Ca2+ release amplitude and timing were enhanced due to the absence of indicator diffusion.
- Observed coactivation of RyR subclusters within single junctions and unique Ca2+ release events.
Conclusions:
- A novel, targeted biosensor allows for precise visualization and measurement of nanodomain Ca2+ dynamics in intact cardiac cells.
- This technology provides mechanistic insights into dyadic RyR function in both healthy hearts and disease states, such as when RyRs become orphaned.
