Multimodal SHG-2PF Imaging of Microdomain Ca2+-Contraction Coupling in Live Cardiac Myocytes
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 and 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 cardiac myocytes.
Objective: To develop a method for imaging sarcomere-level Ca2+-contraction coupling in healthy and disease-model cardiac myocytes.
Methods and Results: Freshly isolated cardiac myocytes 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. Ca2+ signals and sarcomere strain correlated in space and time with short delays. Furthermore, Ca2+ sparks and waves caused contractions in subcellular microdomains, revealing a previously underappreciated role for these events in generating subcellular strain during diastole. Ca2+ activity and sarcomere strain were also imaged in paced cardiomyocytes under mechanical load, revealing spontaneous Ca2+ waves and correlated local contraction in pressure overload-induced cardiac myopathy.
Conclusions: Multi-modal SHG-2PF microscopy enables the simultaneous observation of Ca2+ release and mechanical strain at the sub-sarcomere level in living cardiac myocytes. The method benefits from the label-free nature of SHG, which allows A-bands to be imaged independently of T-tubule morphology and simultaneously with Ca2+ indicators. SHG-2PF imaging is widely applicable to the study of Ca2+-contraction coupling and mechano-chemo-transduction in both health and disease.
- second harmonic generation
- two-photon fluorescence
- multimodality imaging
- calcium sparks
- calcium transients
- Received November 6, 2015.
- Revision received December 2, 2015.
- Accepted December 7, 2015.