Kinetics of Cardiac Thin-Filament Activation Probed by Fluorescence Polarization of Rhodamine-Labeled Troponin C in Skinned Guinea Pig Trabeculae

Abstract

A genetically engineered cardiac TnC mutant labeled at Cys-84 with tetramethylrhodamine-5-iodoacetamide dihydroiodide was passively exchanged for the endogenous form in skinned guinea pig trabeculae. The extent of exchange averaged nearly 70%, quantified by protein microarray of individual trabeculae. The uniformity of its distribution was verified by confocal microscopy. Fluorescence polarization, giving probe angle and its dispersion relative to the fiber long axis, was monitored simultaneously with isometric tension. Probe angle reflects underlying cTnC orientation. In steady-state experiments, rigor cross-bridges and Ca2+ with vanadate to inhibit cross-bridge formation produce a similar change in probe orientation as that observed with cycling cross-bridges (no Vi). Changes in probe angle were found at [Ca2+] well below those required to generate tension. Cross-bridges increased the Ca2+ dependence of angle change (cooperativity). Strong cross-bridge formation enhanced Ca2+ sensitivity and was required for full change in probe position. At submaximal [Ca2+], the thin filament regulatory system may act in a coordinated fashion, with the probe orientation of Ca2+-bound cTnC significantly affected by Ca2+ binding at neighboring regulatory units. The time course of the probe angle change and tension after photolytic release [Ca2+] by laser photolysis of NP-EGTA was Ca2+ sensitive and biphasic: a rapid component approximately 10 times faster than that of tension and a slower rate similar to that of tension. The fast component likely represents steps closely associated with Ca2+ binding to site II of cTnC, whereas the slow component may arise from cross-bridge feedback. These results suggest that the thin filament activation rate does not limit the tension time course in cardiac muscle.

Blebbistatin: use as inhibitor of muscle contraction

“…Hence, Blebbistatin could be used to probe thin-filament Ca 2+ signaling without altering actin-myosin interactions. This property could be utilized in structural studies using light scattering techniques such as X-ray diffraction [18] or with fluorescent probes positioned within the sarcomere by protein exchange techniques [6], provided that Blebbistatin's light sensitivity does not prevent such measurements [32]. Of note, in preliminary studies, we did not find evidence of Blebbistatin inactivation by irradiation with synchrotron-derived X-rays.…”

Insights into the kinetics of Ca2+-regulated contraction and relaxation from myofibril studies

“…The switch kinetics of muscle regulatory proteins have been, with few exceptions [9,17], mostly studied on isolated Tn, its subunits or reconstituted thin filaments [33,37,102,124]. Stopped-flow techniques applied to myofibril suspensions enables comparison The myofibril is mounted within a chamber filled with relaxing solution to the tip of a stiff needle and the tip of an atomic force cantilever.…”

“…This problem can be overcome by loading fibres with photo-labile caged compounds that can rapidly release products, e.g. Ca 2+ or Ca 2+ chelators, by light flashes [4,9,30,69,93,96,100,111,149,156]. However, the use of caged compounds has other drawbacks (discussed in [46,117]).…”

“…For these reasons, skinned fibres had been mostly used to investigate the mechanisms of Ca 2+ -regulated contraction under steady [Ca 2+ ] [16,17,21,28,34,78,120] and only few studies investigated the force kinetics induced by rapid changes in [Ca 2+ ] (e.g. [9,100,111,149]). Several studies that investigate force kinetics in intact and skinned fibres induce force transients by small sinusoidal or step-shaped length changes (e.g.…”

“…These investigations give straightforward insights into cross-bridge kinetics near steady-state conditions but do not include the dynamics of Ca 2+ -induced activation and inactivation of the thin filament that is an integral part of a physiological contraction-relaxation cycle. In contrast to cross-bridge kinetics that have been mainly studied in fibres, the kinetics of thin-filament activation and inactivation have been mainly studied, with few exceptions [9,17] in non-contractile preparations, either with Tn alone or with reconstituted thin filaments (Tn·Tm·actin) interacting with isolated myosin subfragment S1 (e.g. [32,33,37,102,[122][123][124]).…”