Myofilament calcium sensitivity does not affect cross-bridge activation-relaxation kinetics.
We employed single myofibril techniques to test whether the presence of slow skeletal troponin-I (ssTnI) is sufficient to induce increased myofilament calcium sensitivity (EC(50)) and whether modulation of EC(50) affects the dynamics of force development. Studies were performed using rabbit psoas myofibrils activated by rapid solution switch and in which Tn was partially replaced for either recombinant cardiac Tn(cTn) or Tn composed of recombinant cTn-T (cTnT) and cTn-C (cTnC), and recombinant ssTnI (ssTnI-chimera Tn). Tn exchange was performed in rigor solution (0.5 mg/ml Tn; 20 degrees C; 2 h) and confirmed by SDS-PAGE. cTnI exchange induced a decrease in EC(50); ssTnI-chimera Tn exchange induced a further decrease in EC(50) (in microM: endogenous Tn, 1.35 +/- 0.08; cTnI, 1.04 +/- 0.13; ssTnI-chimera Tn, 0.47 +/- 0.03). EC(50) was also decreased by application of 100 microM bepridil (control: 2.04 +/- 0.03 microM; bepridil 1.35 +/- 0.03 microM). Maximum tension was not different between any groups. Despite marked alterations in EC(50), none of the dynamic activation-relaxation parameters were affected under any condition. Our results show that 1) incorporation of ssTnI into the fast skeletal sarcomere is sufficient to induce increased myofilament Ca(2+) sensitivity, and 2) the dynamics of actin-myosin interaction do not correlate with EC(50). This result suggests that intrinsic cross-bridge cycling rate is not altered by the dynamics of thin-filament activation.
de Tombe
Pieter P
PP
Center for Cardiovascular Research, Univ. of Illinois at Chicago, 835 S. Wolcott Avenue, MC901, Chicago IL 60612, USA. pdetombe@uic.edu
Belus
Alexandra
A
Piroddi
Nicoletta
N
Scellini
Beatrice
B
Walker
John S
JS
Martin
Anne F
AF
Tesi
Chiara
C
Poggesi
Corrado
C
D008841:Actin Cytoskeleton / Q000502:physiology*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D007700:Kinetics; D018657:Muscle Fibers, Slow-Twitch / Q000502:physiology; D009126:Muscle Relaxation / Q000502:physiology*; D018482:Muscle, Skeletal / Q000502:physiology*; D009206:Myocardium / Q000378:metabolism; D020033:Protein Isoforms / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019210:Troponin I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D003432:Cross-Linking Reagents; D020033:Protein Isoforms; D011994:Recombinant Proteins; D019210:Troponin I; D002118:Calcium