Cardiac myosin binding protein-C modulates actomyosin binding and kinetics in the in vitro motility assay.
The modulatory role of whole cardiac myosin binding protein-C (cMyBP-C) on myosin force and motion generation was assessed in an in vitro motility assay. The presence of cMyBP-C at an approximate molar ratio of cMyBP-C to whole myosin of 1:2, resulted in a 25% reduction in thin filament velocity (P<0.002) with no effect on relative isometric force under maximally activated conditions (pCa 5). Cardiac MyBP-C was capable of inhibiting actin filament velocity in a concentration-dependent manner using either whole myosin, HMM or S1, indicating that the cMyBP-C does not have to bind to myosin LMM or S2 subdomains to exert its effect. The reduction in velocity by cMyBP-C was independent of changes in ionic strength or excess inorganic phosphate. Co-sedimentation experiments demonstrated S1 binding to actin is reduced as a function of cMyBP-C concentration in the presence of ATP. In contrast, S1 avidly bound to actin in the absence of ATP and limited cMyBP-C binding, indicating that cMyBP-C and S1 compete for actin binding in an ATP-dependent fashion. However, based on the relationship between thin filament velocity and filament length, the cMyBP-C induced reduction in velocity was independent of the number of cross-bridges interacting with the thin filament. In conclusion, the effects of cMyBP-C on velocity and force at both maximal and submaximal activation demonstrate that cMyBP-C does not solely act as a tether between the myosin S2 and LMM subdomains but likely affects both the kinetics and recruitment of myosin cross-bridges through its direct interaction with actin and/or myosin head.
Saber
Walid
W
Department of Medicine, University of Vermont, College of Medicine, Burlington, VT, USA.
Begin
Kelly J
KJ
Department of Medicine, University of Vermont, College of Medicine, Burlington, VT, USA.
Warshaw
David M
DM
Department of Molecular Physiology and Biophysics, University of Vermont, College of Medicine, Burlington, VT, USA.
VanBuren
Peter
P
Department of Medicine, University of Vermont, College of Medicine, Burlington, VT, USA; Department of Molecular Physiology and Biophysics, University of Vermont, College of Medicine, Burlington, VT, USA. Electronic address: vanburen@physiology.med.uvm.edu.
D008841:Actin Cytoskeleton / Q000737:chemistry / Q000378:metabolism; D000205:Actomyosin / Q000737:chemistry* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002474:Cell-Free System / Q000737:chemistry / Q000378:metabolism; D002645:Chickens; D007537:Isometric Contraction / Q000502:physiology; D007700:Kinetics; D018995:Myosin Heavy Chains / Q000737:chemistry / Q000378:metabolism; D018994:Myosin Light Chains / Q000737:chemistry / Q000378:metabolism; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism; D010710:Phosphates; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D000099163:Myosin Binding Protein C
D000205:Actomyosin; D000255:Adenosine Triphosphate; D002352:Carrier Proteins; D018995:Myosin Heavy Chains; D018994:Myosin Light Chains; D015879:Myosin Subfragments; D010710:Phosphates; D000099163:Myosin Binding Protein C