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.

Effects of temperature, ionic strength and pH on the function of skeletal muscle myosin from a eurythermal fish, Fundulus heteroclitus.

The mummichog, Fundulus heteroclitus, is an intertidal fish that exhibits little change in swimming ability despite large and rapid variations in environmental parameters. We therefore tested the hypothesis that this nearly constant function is due to Fundulus myosin being intrinsically insensitive to changes of temperature, ionic strength and pH. In vitro motility assays were used to quantify the speed of unregulated actin filaments on myosin purified from F. heteroclitus glycolytic skeletal muscle. Filament speed was 2.07+/-0.17 microm s(-1) at 26 degrees C, ionic strength (Gamma/2) of 0.08 M Gamma/2 and pH 7.4. Speed increased as temperature increased over the range of 5-36 degrees C with an activation energy (E (a)) of 94.0+/-7.0 kJ mol(-1)) and an enthalpy (DeltaH (double dagger)) of 91.5+/-7.0 kJ mol(-1) at 20 degrees C. A linear relationship between temperature and ATPase activity was also obtained with actin-activated myosin Mg(2+)-ATPase assays over the temperature range 5-35 degrees C with E (a=)59.9+/-2.4 kJ mol(-1) and DeltaH (double dagger)=57.4+/-2.4 kJ mol(-1) at 20 degrees C. There was little or no effect of ionic strength on filament speed over the range 0.19 M Gamma/2-0.54 M Gamma/2. Speed increased significantly at lower ionic strengths and was 7.9-fold higher at 0.08 M Gamma/2 than at 0.19 M Gamma/2. Speed increased with pH with a 16-fold increase between pH 6.7 and 7.4. These results indicate that changes in physiological parameters that include temperature, pH and ionic strength affect the function of unregulated F. heteroclitus myosin, and thus other factors must be responsible for the mummichog's swimming performance being comparatively insensitive to environmental variation.