Proceedings of The Physiological Society
University of Birmingham (2010) Proc Physiol Soc 20, PC20
Investigation of strain response of cardiac muscle by fluorescence lifetime imaging microscopy of the myosin essential light chain
D. S. Ushakov1, C. Mansfield1, V. Caorsi1, M. A. Ferenczi1
1. National Heart & Lung Institute, Imperial College London, London, United Kingdom.
We applied fluorescence lifetime imaging microscopy (FLIM) to map the microenvironment of the myosin essential light chain (ELC) in cardiac and skeletal muscle fibres. Four ELC mutants containing single cysteine residue at different positions in the C-terminal half were labeled with 7-diethylamino-3-((((2-iodoacetamido)ethyl)amino)carbonyl)coumarin. First, the mutants were introduced into permeabilized rabbit psoas muscle fibres under conditions that favour exchange with the native light chain. The fibres were examined under Leica SP5 microscope equipped with a time-correlated single photon counting module. The fluorescence decay in each pixel of FLIM images was fitted with a single exponential and the mean fluorescence lifetime in the A-band regions was found. The mean lifetimes in relaxed fibres were 1.44, 1.64, 1.73 and 1.83 ns, for ELC-142, ELC-127, ELC-160 and ELC-180 respectively. When in rigor, lifetime increased significantly for all label positions, which may be related to a change in conformation of ELC with respect to the heavy chain. However, when 1% stretch was applied to the rigor fibres, the lifetime of ELC-127 and ELC-180 decreased, but did not change in cases of ELC-142 and ELC-160, where the labels are located at the opposite ends of helix F. We applied a similar protocol to exchange ELC mutants in the isolated rat trabeculae. Confocal microscopy confirmed high efficiency and specificity of exchange in the cardiac muscle. The fluorescence lifetime measurements of ELC-180 in relaxed and rigor trabeculae gave similar values to those in psoas fibres, demonstrating the viability of FLIM approach to study the strain response of the cardiac muscle myosin.
Where applicable, experiments conform with Society ethical requirements