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

Circ Res. 2016 Jan 22;118(2):e19-28. doi: 10.1161/CIRCRESAHA.115.307919. Epub 2015 Dec 7.

Abstract

Rationale: Cardiac myocyte contraction is caused by Ca(2+) binding to troponin C, which triggers the cross-bridge power stroke and myofilament sliding in sarcomeres. Synchronized Ca(2+) release causes whole cell contraction and is readily observable with current microscopy techniques. However, it is unknown whether localized Ca(2+) release, such as Ca(2+) sparks and waves, can cause local sarcomere contraction. Contemporary imaging methods fall short of measuring microdomain Ca(2+)-contraction coupling in live cardiac myocytes.

Objective: To develop a method for imaging sarcomere level Ca(2+)-contraction coupling in healthy and disease model cardiac myocytes.

Methods and results: Freshly isolated cardiac myocytes were loaded with the Ca(2+)-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. Ca(2+) signals and sarcomere strain correlated in space and time with short delays. Furthermore, Ca(2+) sparks and waves caused contractions in subcellular microdomains, revealing a previously underappreciated role for these events in generating subcellular strain during diastole. Ca(2+) activity and sarcomere strain were also imaged in paced cardiac myocytes under mechanical load, revealing spontaneous Ca(2+) waves and correlated local contraction in pressure-overload-induced cardiomyopathy.

Conclusions: Multimodal second harmonic generation 2-photon fluorescence microscopy enables the simultaneous observation of Ca(2+) 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 Ca(2+) indicators. Second harmonic generation 2-photon fluorescence imaging is widely applicable to the study of Ca(2+)-contraction coupling and mechanochemotransduction in both health and disease.

Keywords: calcium signaling; cardiomyopathies; mechanotransduction, cellular; microscopy, fluorescence, multiphoton; multimodal imaging; myocardial contraction; sarcomeres.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Review

MeSH terms

  • Aniline Compounds
  • Animals
  • Cardiomyopathies / metabolism*
  • Cardiomyopathies / physiopathology
  • Disease Models, Animal
  • Excitation Contraction Coupling*
  • Fluorescent Dyes
  • Kinetics
  • Male
  • Mechanotransduction, Cellular
  • Membrane Microdomains / metabolism*
  • Mice
  • Microscopy, Confocal*
  • Microscopy, Fluorescence, Multiphoton*
  • Multimodal Imaging / methods*
  • Myocardial Contraction*
  • Myocytes, Cardiac / metabolism*
  • Rats, Sprague-Dawley
  • Sarcomeres / metabolism*
  • Stress, Mechanical
  • Xanthenes

Substances

  • Aniline Compounds
  • Fluo 4
  • Fluorescent Dyes
  • Xanthenes