RATIONALE:: Cardiomyocyte contraction is caused by Ca binding to troponin C, which triggers the cross-bridge power stroke and myofilament sliding in sarcomeres. Synchronized Ca release causes whole cell contraction and is readily observable with current microscopy techniques. However, it is unknown whether localized Ca release, such as Ca sparks and waves, can cause local sarcomere contraction. Contemporary imaging methods fall short of measuring microdomain Ca-contraction coupling in live cardiac myocytes. OBJECTIVE:: To develop a method for imaging sarcomere-level Ca-contraction coupling in healthy and disease-model cardiac myocytes. METHODS AND RESULTS:: Freshly isolated cardiac myocytes were loaded with the Ca-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. Ca signals and sarcomere strain correlated in space and time with short delays. Furthermore, Ca sparks and waves caused contractions in subcellular microdomains, revealing a previously underappreciated role for these events in generating subcellular strain during diastole. Ca activity and sarcomere strain were also imaged in paced cardiomyocytes under mechanical load, revealing spontaneous Ca waves and correlated local contraction in pressure overload-induced cardiac myopathy. CONCLUSIONS:: Multi-modal SHG-2PF microscopy enables the simultaneous observation of Ca 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 Ca indicators. SHG-2PF imaging is widely applicable to the study of Ca-contraction coupling and mechano-chemo-transduction in both health and disease.
ASJC Scopus subject areas
- Cardiology and Cardiovascular Medicine