Abstract
The muscle types present with variable fatigue tolerance, in part due to the myosin isoform expressed. However, the critical steps that define “fatigability” in vivo of fast vs. slow myosin isoforms, at the molecular level, are not yet fully understood. We examined the modulation of the ATP-induced myosin subfragment 1 (S1) dissociation from pyrene-actin by inorganic phosphate (Pi), pH, and temperature using a specially modified stopped-flow system that allowed fast kinetics measurements at physiological temperature. We contrasted the properties of rabbit psoas (fast) and bovine masseter (slow) myosins (obtained from samples collected from New Zealand rabbits and from a licensed abattoir, respectively, according to institutional and national ethics permits). To identify ATP cycling biochemical intermediates, we assessed ATP binding to a preequilibrated mixture of actomyosin and variable [ADP], pH (pH 7 vs. pH 6.2), and Pi (zero, 15, or 30 added mM Pi) in a range of temperatures (5 to 45°C). Temperature and pH variations had little, if any, effect on the ADP dissociation constant (KADP) for fast S1, but for slow S1, KADP was weakened with increasing temperature or low pH. In the absence of ADP, the dissociation constant for phosphate (KPi) was weakened with increasing temperature for fast S1. In the presence of ADP, myosin type differences were revealed at the apparent phosphate affinity, depending on pH and temperature. Overall, the newly revealed kinetic differences between myosin types could help explain the in vivo observed muscle type functional differences at rest and during fatigue.
Highlights
MYOSIN II exists in multiple isoforms [49], with slow muscles expressing myosin heavy chain type 1 (MyHC-1 known as MyHC-) and fast muscles expressing one or more of myosin heavy chain type 2 myosins (MyHC-2a, 2b, or 2x)
These two muscle types yield essentially pure MyHC isoform (e.g., Refs. 1, 25) for rabbit psoas and [55] for bovine masseter, a result confirmed in routine SDS-PAGE by us and others and by the expected value of KI for ADP (KADP) which is characteristic of a pure MyHC isoform
When actin.PsoS1 and actin.MassS1 were mixed with ATP, as shown in Fig. 2, A and B, the observed stopped-flow transients were described by a single exponential for both myosin isoforms (Fig. 2, A and B)
Summary
MYOSIN II exists in multiple isoforms [49], with slow muscles expressing myosin heavy chain type 1 (MyHC-1 known as MyHC-) and fast muscles expressing one or more of myosin heavy chain type 2 myosins (MyHC-2a, 2b, or 2x). The study of kinetics of the actomyosin (A.M) interaction cycle identifies clear intermediate steps (for a review see Ref. 5) Such studies have revealed that slow skeletal myosin heavy chain isoforms (MyHC 1) have distinct properties from fast isoforms (MyHC 2s), e.g., regarding ATPase activity and the rate and equilibrium constants of the various biochemical steps, which are expected to dictate their different mechanical properties. The coupling of biochemical steps with mechanical events has, not been fully elucidated [22], while the “laws” governing how ensembles of myosins integrate within the organized sarcomere [18, 19, 40] are not yet fully defined; this can be attributed partly to lack of physiologically relevant experimental evidence at the molecular level This is especially true on the question of muscle fatigue, a complex multifaceted phenomenon. For the purposes of this work, fatigue is considered in the context of factors influencing the actomyosin cycle in a way to cause slowing of the cycle and/or weaker actomyosin interactions
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