Functional diversity among a family of human skeletal muscle myosin motors

Abstract

Human skeletal muscle fibers express five highly conserved type-II myosin heavy chain (MyHC) genes in distinct spatial and temporal patterns. In addition, the human genome contains an intact sixth gene, MyHC-IIb, which is thought under most circumstances not to be expressed. The physiological and biochemical properties of individual muscle fibers correlate with the predominantly expressed MyHC isoform, but a functional analysis of homogenous skeletal muscle myosin isoforms has not been possible. This is due to the difficulties of separating the multiple isoforms usually coexpressed in muscle fibers, as well as the lack of an expression system that produces active recombinant type II skeletal muscle myosin. In this study we describe a mammalian muscle cell expression system and the functional analysis of all six recombinant human type II skeletal muscle myosin isoforms. The diverse biochemical activities and actin-filament velocities of these myosins indicate that they likely have distinct functions in muscle. Our data also show that ATPase activity and motility are generally correlated for human skeletal muscle myosins. The exception, MyHC-IIb, encodes a protein that is high in ATPase activity but slow in motility; this is the first functional analysis of the protein from this gene. In addition, the developmental isoforms, hypothesized to have low ATPase activity, were indistinguishable from adult-fast MyHC-IIa and the specialized MyHC-Extraocular isoform, that was predicted to be the fastest of all six isoforms but was functionally similar to the slower isoforms.

Molecular consequences of the R453C hypertrophic cardiomyopathy mutation on human β-cardiac myosin motor function

“…β-cardiac myosin in heterologous expression systems, possibly due to the lack of muscle-cell-specific chaperones (11). The ∼30% decreases that we observed in both the k cat of the ATPase and the unloaded maximum velocity of movement of the R453C mutant S1 compared with the WT S1 result in corresponding increases in both t c and t s (Fig.…”

“…The myosin proteins used in this study were constructed and produced as previously described (11,27), using the AdEasy Vector System (Qbiogene, Inc) with minor modifications. MHY7 cDNA and myosin light chain 3 (MYL3) (ventricular ELC) were purchased from Open Biosystems (Thermo).…”

“…Additionally, cardiac biopsies yield limited amounts of enzymatically active motor and are heterogeneous in the proportion of the mutated form. This problem has now been addressed by a recently developed muscle cell expression system that provides a source of active recombinant human β-cardiac muscle myosin (11).…”

Mechanistic Heterogeneity in Contractile Properties of α-Tropomyosin (TPM1) Mutants Associated with Inherited Cardiomyopathies

“…Actin was purified from chicken pectoral muscle acetone powder (38). Human ␤-cardiac myosin subfragment 1 (S1), containing the ATPase head domain and the essential light chain, was expressed and purified from C2C12 cells (33,39). Representative purifications and reconstitution into regulated thin filaments (RTFs) (35)(36)(37) Labeling of TnC 3 with ANS and NBD-TnC 3 was purified as described above and dialyzed into 50 mM Tris, 100 mM KCl, 1 mM EGTA, pH 7.5, at 4°C.…”

The role of mechanics in biological and bio-inspired systems

“…Muscle formation begins with a process of myosin molecules forming bipolar filaments. Myosin isoforms have varied molecular structures that influence their force generation, as well as energy usage rates that inform contractile functionality 12,13 . The aggregation of these bipolar filaments into sarcomeres forms the basis of muscles, and the system loops back as environmental feedback from muscle activation influences which myosins are recruited.…”