Cardiac myosin isoforms exhibit differential rates of MgADP release and MgATP binding detected by myocardial viscoelasticity

Abstract

We measured myosin crossbridge detachment rate and the rates of MgADP release and MgATP binding in mouse and rat myocardial strips bearing one of the two cardiac myosin heavy chain (MyHC) isoforms. Mice and rats were fed an iodine-deficient, propylthiouracil diet resulting in ~100% expression of β-MyHC in the ventricles. Ventricles of control animals expressed ~100% α-MyHC. Chemically-skinned myocardial strips prepared from papillary muscle were subjected to sinusoidal length perturbation analysis at maximum calcium activation pCa 4.8 and 17°C. Frequency characteristics of myocardial viscoelasticity were used to calculate crossbridge detachment rate over 0.01 to 5 mM [MgATP]. The rate of MgADP release, equivalent to the asymptotic value of crossbridge detachment rate at high MgATP, was highest in mouse α-MyHC (111.4±6.2 s−1) followed by rat α-MyHC (65.0±7.3 s−1), mouse β-MyHC (24.3±1.8 s−1) and rat β-MyHC (15.5±0.8 s−1). The rate of MgATP binding was highest in mouse α-MyHC (325±32 mM−1.s−1) then mouse β-MyHC (152±23 mM−1.s−1), rat α-MyHC (108±10 mM−1.s−1) and rat β-MyHC (55±6 mM−1.s−1). Because the events of MgADP release and MgATP binding occur in a post power-stroke state of the myosin crossbridge, we infer that MgATP release and MgATP binding must be regulated by isoform- and species-specific structural differences located outside the nucleotide binding pocket, which is identical in sequence for these four myosins. We postulate that differences in the stiffness profile of the entire myosin molecule, including the thick filament and the myosin-actin interface, are primarily responsible for determining the strain on the nucleotide binding pocket and the subsequent differences in the rates of nucleotide release and binding observed among the four myosins examined here.

Transgenic Mouse α- and β-Cardiac Myosins Containing the R403Q Mutation Show Isoform-dependent Transient Kinetic Differences

“…Remarkably, a 4 -5-fold difference in rates of cross-bridge detachment at high ATP (from which the ADP release rate can be obtained) was measured mechanically for ␣-and ␤-cardiac myosin isoforms in skinned mouse myocardial strips (17). The in vitro motility assay, a simplified model for actomyosin interactions, has consistently shown a 2-fold difference in velocities between the isolated cardiac myosin isoforms of individual species (18,19).…”

Effects of a myofilament calcium sensitizer on left ventricular systolic and diastolic function in rats with volume overload heart failure

“…cTnI regulates thin-filament function, and Ser-23/24 phosphorylation by PKA decreases Ca 2ϩ sensitivity and myofilament cross-bridge cycling kinetics (31,36) and increases the rate of cTnC-Ca 2ϩ dissociation (23). Finally, chemically skinned myocardium containing primarily ␣-MHC has a higher ATPase rate, faster sarcomeric shortening velocity, faster rate of tension relaxation, higher power production, and greater rate of force development compared with ␤-MHC (11,39). Taken together, we suggest that there is a complex interrelationship among these three proteins, and the relative importance of each in our model depends on hemodynamic load as well as presence or absence of the myofilament Ca 2ϩ sensitizer.…”

Magnesium Modulates Actin Binding and ADP Release in Myosin Motors

“…Cardiac papillary muscle was taken from one male conventional swine (E. M. Parsons) weighing 16 kg. Myocardial strips were dissected further, chemically skinned, and studied at 27°C as described previously (39). Free Mg 2ϩ concentrations of 1, 2, 4, and 8 mM were applied at pCa 4.8 by exchanging volumes of 1 or 8 mM free Mg 2ϩ solutions.…”

“…The rate 2c is the equivalent of the myosin cross-bridge detachment rate, and its reciprocal is equal to time on, and the average time myosin is attached to actin (39).…”