Ca2+ sensitivity of regulated cardiac thin filament sliding does not depend on myosin isoform.
Myosin heavy chain (MHC) isoforms in vertebrate striated muscles are distinguished functionally by differences in chemomechanical kinetics. These kinetic differences may influence the cross-bridge-dependent co-operativity of thin filament Ca(2+) activation. To determine whether Ca(2+) sensitivity of unloaded thin filament sliding depends upon MHC isoform kinetics, we performed in vitro motility assays with rabbit skeletal heavy meromyosin (rsHMM) or porcine cardiac myosin (pcMyosin). Regulated thin filaments were reconstituted with recombinant human cardiac troponin (rhcTn) and alpha-tropomyosin (rhcTm) expressed in Escherichia coli. All three subunits of rhcTn were coexpressed as a functional complex using a novel construct with a glutathione S-transferase (GST) affinity tag at the N-terminus of human cardiac troponin T (hcTnT) and an intervening tobacco etch virus (TEV) protease site that allows purification of rhcTn without denaturation, and removal of the GST tag without proteolysis of rhcTn subunits. Use of this highly purified rhcTn in our motility studies resulted in a clear definition of the regulated motility profile for both fast and slow MHC isoforms. Maximum sliding speed (pCa 5) of regulated thin filaments was roughly fivefold faster with rsHMM compared with pcMyosin, although speed was increased by 1.6- to 1.9-fold for regulated over unregulated actin with both MHC isoforms. The Ca(2+) sensitivity of regulated thin filament sliding speed was unaffected by MHC isoform. Our motility results suggest that the cellular changes in isoform expression that result in regulation of myosin kinetics can occur independently of changes that influence thin filament Ca(2+) sensitivity.
Schoffstall
Brenda
B
Institute of Molecular Biophysics, Department of Biological Science, Bio Unit One, Florida State University, Tallahassee, FL 32306-4370, USA.
Brunet
Nicolas M
NM
Williams
Shanedah
S
Miller
Victor F
VF
Barnes
Alyson T
AT
Wang
Fang
F
Compton
Lisa A
LA
McFadden
Lori A
LA
Taylor
Dianne W
DW
Seavy
Margaret
M
Dhanarajan
Rani
R
Chase
P Bryant
PB
D008841:Actin Cytoskeleton / Q000502:physiology*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D024722:Cardiac Myosins / Q000502:physiology; D006321:Heart / Q000502:physiology*; D006801:Humans; D007527:Isoenzymes / Q000502:physiology; D007700:Kinetics; D009200:Myocardial Contraction / Q000502:physiology*; D009206:Myocardium / Q000378:metabolism*; D015879:Myosin Subfragments / Q000502:physiology; D009218:Myosins / Q000502:physiology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D013552:Swine; D014335:Tropomyosin / Q000378:metabolism; D014336:Troponin / Q000378:metabolism; D020107:Troponin T / Q000378:metabolism
D007527:Isoenzymes; D015879:Myosin Subfragments; D011994:Recombinant Proteins; D014335:Tropomyosin; D014336:Troponin; D020107:Troponin T; D024722:Cardiac Myosins; D009218:Myosins; D002118:Calcium