| Autor: |
Ziliang Mao, Zhong Jian, Ye Chen-Izu, James W. Chan, Samir Awasti, Leighton T. Izu |
| Rok vydání: |
2016 |
| Předmět: |
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| Zdroj: |
Biophysical Journal. 110:432a |
| ISSN: |
0006-3495 |
| Popis: |
Rationale: Cardiomyocyte contraction is caused by Ca2+ binding to troponin C, which triggers the cross-bridge power stroke and myofilament sliding in sarcomeres. Synchronized Ca2+ release causes whole cell contraction, which is readily observable with current microscopy techniques. However, it is unknown whether localized Ca2+ release, such as Ca2+ sparks and waves, can cause local sarcomere contraction. Contemporary imaging methods fall short of measuring microdomain Ca2+-contraction coupling in live cardiomyocytes.Objective: To develop a method for imaging sarcomere-level Ca2+-contraction coupling in live cardiomyocytes at high spatiotemporal resolution.Methods and Results: Freshly isolated cardiomyocytes were loaded with the Ca2+-indicator Fluo-4. A confocal microscope equipped with a femtosecond-pulsed near-infrared laser was used to simultaneously excite second harmonic generation (SHG) from A-bands of myofibrils and two-photon fluorescence (2PF) from Fluo-4. Analysis methods and algorithms were developed for tracking A-band displacement and computing sarcomere strain maps. We found that Ca2+ signals and sarcomere strain correlated in space and time with short delays; importantly, localized Ca2+ sparks and waves caused non-uniform sarcomere contractions in subcellular microdomains.Conclusion: Multi-modal SHG-2PF microscopy enables the study of local mechanical strain in relation to Ca2+ sparks and waves which are often associated with pathological conditions. The method has multiple advantages: (1) SHG is a label-free process that requires no dyes or genetic labeling; (2) It images A-bands independently of T-tubule morphology; (3) It enables simultaneous measurements of microdomain contraction and local Ca2+ levels. Therefore SHG-2PF imaging is widely applicable in studying local Ca2+and sub-sarcomere contraction coupling and the mechano-chemo-transduction in live cardiomyocytes. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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