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

Abstract

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 (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 cardiac myocytes under mechanical load, revealing spontaneous Ca2+ waves and correlated local contraction in pressure overload-induced cardiomyopathy. 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.

ORAI1, STIM1/2, and RYR1 shape subsecond Ca 2+ microdomains upon T cell activation

“…outside the transverse and axial tubular system (16). Using second harmonic generation and 2-photon-fluorescence, Ca 2+ microdomains generated by RYR2 as well as local sarcomere contractions were analyzed, resulting in detection of locally coordinated Ca 2+ sparks and contractions (17). Although the ER in T cells is not as much ordered as the sarcoplasmic reticulum in cardiac myocytes, we observed alternating patches of RYR and ORAI1 at or very close below the plasma membrane (Fig 4G–J), indicating that proper localization of RYR may in general be an important mechanism for regulation of Ca 2+ microdomains.…”

Mechano‐chemo‐transduction in cardiac myocytes

“…However, the MCT also caused spontaneous Ca 2+ sparks during diastole that sometimes arise synchronously to form Ca 2+ waves (Awasthi et al . ), which could trigger afterdepolarizations and premature action potentials that provide substrate for cardiac arrhythmias (Kass & Tsien, ; Berlin et al . ; Katra & Laurita, ).…”

Porphyrin Dyes for Nonlinear Optical Imaging of Live Cells

“…Multimodal imaging harnesses the advantages of several imaging techniques to visualize discrete biological processes simultaneously, which otherwise would not be possible by using just one technique at a time ( Awasthi et al., 2016 , Cheng et al., 2011 , Nuriya et al., 2016 , Weissleder and Pittet, 2008 ). TPEF and SHG-based multimodal imaging is mostly restricted to the situation where part of the sample itself generates SHG signals, for example, sarcomeres in cardiomyocytes, thus requiring only a single dye to be used for fluorescence ( Awasthi et al., 2016 ). We performed TPEF and SHG-based multimodal imaging of far-red to NIR emitting dye AK-1 in HEK293T cells with two fluorescent cell trackers, mitochondrial tracker RH123 and LysoTracker Yellow HCK-123 ( Figure 5 ).…”