Temperature-dependence of isometric tension and cross-bridge kinetics of cardiac muscle fibers reconstituted with a tropomyosin internal deletion mutant

¹ Univ of Iowa
² Univ Illinois at Chicago

^* To whom correspondence should be addressed. E-mail: masataka-kawai{at}uiowa.edu.

Submitted on March 7, 2006
Revised on April 4, 2006
Accepted on 30 August 2006

	Abstract

The effect of temperature on isometric tension and cross-bridge kinetics was studied with a tropomyosin (Tm) internal deletion mutant AS-23Tm [Ala-Ser-Tm (47-123): Landis et al, J Biol Chem 274, 31279, 1999] in bovine cardiac muscle fibers by using the thin filament extraction and reconstitution technique. The results are compared with those from actin reconstituted alone, cardiac muscle-derived control acetyl-Tm, and recombinant control AS-Tm. In all four reconstituted muscle groups, isometric tension and stiffness increased linearly with temperature in the range 5-40°C for fibers activated in the presence of saturating ATP and Ca²⁺. The slopes of the temperature-tension plots of the two controls were very similar, whereas the slope derived from fibers with actin alone had ~ 40% the control value, and the slope from mutant Tm had ~ 36% the control value. Sinusoidal analysis was performed to study the temperature dependence of cross-bridge kinetics. All three exponential processes A, B, and C were identified in the high temperature range (30-40°C), only processes B and C were identified in the mid temperature range (15-25°C), and only process C was identified in the low temperature range (5-10°C). At a given temperature, similar apparent rate constants (2a, 2b, 2c) were observed in all four muscle groups, whereas their magnitudes were markedly less in the order of AS-23Tm < Actin < AS-Tm Acetyl-Tm groups. Our observations are consistent with the hypothesis that Tm enhances hydrophobic and stereospecific interactions (positive allosteric effect) between actin and myosin, but 23Tm decreases these interactions (negative allosteric effect). Our observations further indicate that tension/cross-bridge is increased by Tm, but is diminished by 23Tm. We conclude that Tm affects the conformation of actin so as to increase the area of hydrophobic interaction between actin and myosin molecules.

Key Words: Susoidal analysis, apparent rate constant, cross-bridge kinetics, hydrophobic interaction, thermodynamic parameters, thin filament

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