Kinetics of cardiac thin-filament activation probed by fluorescence polarization of rhodamine-labeled troponin C in skinned guinea pig trabeculae

¹ Philadelphia College of Osteopathic Medicine
² Thomas Jefferson University
³ Drexel University College of Medicine
⁴ University of Washington

^* To whom correspondence should be addressed. E-mail: robert.barsotti{at}jefferson.edu.

Submitted on August 18, 2005
Revised on September 26, 2005
Accepted on 11 October 2005

	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 trabecula. The uniformity of its distribution was verified by confocal microscopy. Fluorescence polarization (FP) 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 following photolytic release [Ca2+] by laser photolysis of NP-EGTA was Ca2+ sensitive and biphasic: a rapid component ~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, while 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.

Key Words: NP-EGTA, fluoresence polarization, kinetics, laser photolysis, thin filament regulation, troponin c

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