What limits the velocity of fast-skeletal muscle contraction in mammals?
In rat skeletal muscle the unloaded shortening velocity (Vo) is defined by the myosin isoform expressed in the muscle fibre. In 2001 we suggested that ADP release from actomyosin in solution (controlled by k(-AD)) was of the right size to limit Vo. However, to compare mechanical and solution kinetic data required a series of corrections to compensate for the differences in experimental conditions (0.5 M KCl, 22 degrees C for kinetic assays of myosin, 200 mM ionic strength, 12 degrees C to measure Vo). Here, a method was developed to prepare heavy meromyosin (HMM) from pure myosin isoforms isolated from single muscle fibres and to study k(-AD) (determined from the affinity of the acto-myosin complex for ADP, KAD) and the rate of ATP-induced acto-HMM dissociation (controlled by K1k+2) under the same experimental condition used to measure Vo). In fast-muscle myosin isolated from a wide range of mammalian muscles, k(-AD) was found to be too fast to limit Vo, whereas K1k+2 was of the right magnitude for ATP-induced dissociation of the cross-bridge to limit shortening velocity. The result was unexpected and prompted further experiments using the stopped-flow approach on myosin subfragment-1 (S1) and HMM obtained from bulk preparations of rabbit and rat muscle. These confirmed that the rate of cross-bridge dissociation by ATP limits the velocity of contraction for fast myosin II isoforms at 12 degrees C, while k(-AD) limits the velocity of slow myosin II isoforms. Extrapolating our data to 37 degrees C suggests that at physiological temperature the rate of ADP dissociation may limit Vo for both isoforms.
Nyitrai
Miklós
M
Department of Biophysics, University of Pécs, Pecs, Hungary.
Rossi
Rosetta
R
Adamek
Nancy
N
Pellegrino
Maria Antonietta
MA
Bottinelli
Roberto
R
Geeves
Michael A
MA
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D008954:Models, Biological; D009119:Muscle Contraction / Q000502:physiology*; D018656:Muscle Fibers, Fast-Twitch / Q000502:physiology*; D018482:Muscle, Skeletal / Q000502:physiology*; D015879:Myosin Subfragments / Q000502:physiology*; D024462:Myosin Type II / Q000502:physiology; D009994:Osmolar Concentration; D020033:Protein Isoforms / Q000502:physiology; D011817:Rabbits; D051381:Rats; D013552:Swine; D013696:Temperature
D015879:Myosin Subfragments; D020033:Protein Isoforms; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D024462:Myosin Type II