Date Published: September 18, 2018
Publisher: Public Library of Science
Author(s): Lien A. Phung, Sira M. Karvinen, Brett A. Colson, David D. Thomas, Dawn A. Lowe, Guillermo López Lluch.
The recent discovery that myosin has two distinct states in relaxed muscle–disordered relaxed (DRX) and super-relaxed (SRX)–provides another factor to consider in our fundamental understanding of the aging mechanism in skeletal muscle, since myosin is thought to be a potential contributor to dynapenia (age-associated loss of muscle strength independent of atrophy). The primary goal of this study was to determine the effects of age on DRX and SRX states and to examine their sex specificity. We have used quantitative fluorescence microscopy of the fluorescent nucleotide analog 2′/3′-O-(N-methylanthraniloyl) ATP (mantATP) to measure single-nucleotide turnover kinetics of myosin in skinned skeletal muscle fibers under relaxing conditions. We examined changes in DRX and SRX in response to the natural aging process by measuring the turnover of mantATP in skinned fibers isolated from psoas muscle of adult young (3–4 months old) and aged (26–28 months old) C57BL/6 female and male mice. Fluorescence decays were fitted to a multi-exponential decay function to determine both the time constants and mole fractions of fast and slow turnover populations, and significance was analyzed by a t-test. We found that in females, both the DRX and SRX lifetimes of myosin ATP turnover at steady state were shorter in aged muscle fibers compared to young muscle fibers (p ≤ 0.033). However, there was no significant difference in relaxation lifetime of either DRX (p = 0.202) or SRX (p = 0.804) between young and aged male mice. No significant effects were measured on the mole fractions (populations) of these states, as a function of sex or age (females, p = 0.100; males, p = 0.929). The effect of age on the order of myosin heads at rest and their ATPase function is sex specific, affecting only females. These findings provide new insight into the molecular factors and mechanisms that contribute to aging muscle dysfunction in a sex-specific manner.
In muscle, myosin uses energy from ATP hydrolysis to perform mechanical work with Ca2+ activated actin during contraction. However, when myosin is saturated with ATP in the absence of Ca2+, myosin head domains are prevented from associating with actin, so the muscle remains relaxed. Recent work by Cooke and others has shown that there are two distinct states of myosin in this relaxed condition [1–3] (Fig 1). The biochemical features of the two states were measured using a nucleotide chase experiment on skinned skeletal muscle fibers, showing that myosin single-nucleotide turnover is a multi-phasic process [1, 4]. The first relaxed state is disordered relaxed (DRX), in which myosin heads are free from each other and the thick filament backbone but do not interact with actin, resulting in orientational disorder [5, 6]. The lifetime of ATP turnover by skeletal muscle DRX myosin is less than 30 seconds (s), similar to that measured for ATPase kinetics of purified myosin [2, 7, 8]. The second relaxed state, designated super-relaxed (SRX), is proposed to be related to the highly ordered interacting head motif observed by X-ray diffraction, electron microscopy, and fluorescence polarization [3, 9–11]. In the SRX state, myosin binds ATP with extremely slow turnover lifetime (>100 s), and the two heads are proposed to be ordered in an auto-inhibitory complex. In vitro, relaxed fast skeletal muscle fibers typically contain ~70% DRX and ~30% SRX myosin [1, 2]. The SRX state is emerging as an important player in the regulation of muscle contraction and is helping address decades-old discrepancies in the myosin field. Specifically, the high rate of ATP turnover by purified myosin in solution relative to ~10-fold slower ATP turnover in muscle fibers is explained by the decreased SRX content in purified myosin [7, 8, 12, 13].
Statistical analyses were carried out using IBM SPSS for Windows 23 statistical software (SPSS Inc., Chicago, IL, USA). The Shapiro-Wilk test was used to investigate within group normality for a given parameter of interest. Levene’s test was conducted to assess the homogeneity of the variance assumption. T-tests were used to analyze data and P-values less than 0.05 were considered statistically significant.
This study examined the impact of the natural aging process on relaxed states of myosin in skeletal muscle fibers from male and female mice. Age-related alterations in myosin function during contraction and relaxation have been reported [15, 16, 42–47], but ours is the first to report findings for the changes in myosin ATPase activity in the DRX and SRX states with age and sex specificity. Following our previous work that showed altered SRX under relaxed conditions in fibers from an ovariectomy-induced aging model , here we measured the single-nucleotide turnover in resting skeletal muscle fibers isolated from naturally aged mice from both sexes. This study was done on chemically skinned fibers in fully relaxed conditions, thus the measured ATPase activity reflects mainly the work from myosin without actin activation or membrane ATPase proteins.
In conclusion, we have shown that the rate of myosin nucleotide turnover during relaxation is affected by age in a sex-specific manner: Age increases basal ATP turnover in muscles of female but not male mice. Together with previous results, our findings further indicate that myosin is a likely molecular contributor to dynapenia. Further studies are needed to better understand the molecular mechanisms by which myosin, its associated sarcomeric proteins, and estradiol signaling contribute to the progression of muscle weakness.