Multimodal SHG-2PF Imaging of Microdomain Ca2+-Contraction Coupling in Live Cardiac Myocytes

Rationale: Cardiac myocyte 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 from A-bands of myofibrils and 2-photon fluorescence 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 cardiac myocytes under mechanical load, revealing spontaneous Ca2+ waves and correlated local contraction in pressure-overload-induced cardiomyopathy. Conclusions: Multimodal second harmonic generation 2-photon fluorescence microscopy enables the simultaneous observation of Ca2+ 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 Ca2+ indicators. Second harmonic generation 2-photon fluorescence imaging is widely applicable to the study of Ca2+-contraction coupling and mechanochemotransduction in both health and disease.