Multimodal SHG-2PF Imaging of Microdomain Ca 2+ -Contraction Coupling in Live Cardiac Myocytes
| Autor: | Nipavan Chiamvimonvat, Zhong Jian, Brent M. Wood, Ziliang Mao, Dennis L Matthews, Deborah K. Lieu, Julie Bossuyt, Aaron Lerner, Leighton T. Izu, Rahwa A Woldeyesus, Kit S. Lam, Samir Awasthi, Yi-Je Chen, Ye Chen-Izu, Rafael Shimkunas, James W. Chan, Trevor Landas |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
Male
0301 basic medicine Fluorescence-lifetime imaging microscopy Myofilament Mechanotransduction Physiology Analytical chemistry multimodal imaging Cardiorespiratory Medicine and Haematology Cardiovascular Sarcomere Troponin C Mice Fluorescence microscope Myocyte Excitation Contraction Coupling Physics Microscopy Aniline Compounds Cardiac myocyte Heart Disease Confocal Cardiology and Cardiovascular Medicine Cardiac Sarcomeres cardiomyopathies multiphoton Clinical Sciences Bioengineering Stress calcium signaling Fluorescence 03 medical and health sciences Membrane Microdomains Animals Fluorescent Dyes Myocytes Animal Mechanical Myocardial Contraction Rats Kinetics 030104 developmental biology Xanthenes Cardiovascular System & Hematology Disease Models Biophysics Sprague-Dawley Cellular Myofibril |
| Zdroj: | Circulation research, vol 118, iss 2 |
| ISSN: | 1524-4571 0009-7330 |
| Popis: | Rationale: Cardiac myocyte contraction is caused by Ca 2+ binding to troponin C, which triggers the cross-bridge power stroke and myofilament sliding in sarcomeres. Synchronized Ca 2+ release causes whole cell contraction and is readily observable with current microscopy techniques. However, it is unknown whether localized Ca 2+ release, such as Ca 2+ sparks and waves, can cause local sarcomere contraction. Contemporary imaging methods fall short of measuring microdomain Ca 2+ -contraction coupling in live cardiac myocytes. Objective: To develop a method for imaging sarcomere level Ca 2+ -contraction coupling in healthy and disease model cardiac myocytes. Methods and Results: Freshly isolated cardiac myocytes were loaded with the Ca 2+ -indicator fluo-4. A confocal microscope equipped with a femtosecond-pulsed near-infrared laser was used to simultaneously excite second harmonic generation from A-bands of myofibrils and 2-photon fluorescence from fluo-4. Ca 2+ signals and sarcomere strain correlated in space and time with short delays. Furthermore, Ca 2+ sparks and waves caused contractions in subcellular microdomains, revealing a previously underappreciated role for these events in generating subcellular strain during diastole. Ca 2+ activity and sarcomere strain were also imaged in paced cardiac myocytes under mechanical load, revealing spontaneous Ca 2+ waves and correlated local contraction in pressure-overload–induced cardiomyopathy. Conclusions: Multimodal second harmonic generation 2-photon fluorescence microscopy enables the simultaneous observation of Ca 2+ release and mechanical strain at the subsarcomere level in living cardiac myocytes. The method benefits from the label-free nature of second harmonic generation, which allows A-bands to be imaged independently of T-tubule morphology and simultaneously with Ca 2+ indicators. Second harmonic generation 2-photon fluorescence imaging is widely applicable to the study of Ca 2+ -contraction coupling and mechanochemotransduction in both health and disease. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|