A novel single skeletal muscle cell in vitro motility assay : Effects of aging and non-enzymatic glycosylation on myosin function

Abstract: The aims of this study were to develop a single skeletal muscle fiber in vitro motility assay, and to employ the assay in order to study the effects of aging and non-enzymatic glycosylation on the mechanical properties of the motor protein myosin in an attempt to improve our understanding of the molecular mechanisms underlying the aging-related impairment in regulation of muscle function. Myosin was extracted from a millimeter short single muscle fiber segment directly onto a nitrocellulosecoated coverslip for subsequent analyses of the functional properties of the motor protein. Approximately 80% of the myosin heavy chain (MyHC), myosin light chains (MyLCs) and myosin-binding protein C (MyBP-C) was extracted from the cell segment, while no significant extraction of actin and actin-associated proteins was observed. The isolated and immobilized myosin formed a narrow, dense streak on which fluorescent-labeled actin filaments were propelled in a smooth and bi-directional motion, and with small variability in motility speed. Actin filament speed was highly dependent on the MyHC isoform extracted, i.e., a significant difference was observed at 25 deg. C between rat beta/slow (type 1) (1.31 ± 0.23 µm/s, n=11) and IIXB (5.81 ± 0.35 µm/s, n=6), or IIB (6.07 ± 0.33 µm/s, n=8) MyHC isoforms, and between human MyHC isoforms of the type 1 (0.69 ± 0.15 µm/s, n=11), type IIA (2.63 ± 0.34 µm/s, n=7) and IIAX (3.48 ± 0.69 µm/s, n=3). In vitro motility speed showed a high correlation with maximum velocity of unloaded shortening (V0) in slow and fast human single fibers and in rat fibers expressing fast myosin isoforms, indicating that actin filament speed is a good molecular analogue to contractile speed at the fiber level. Motility speed measurements in the 10-350C temperature range were accomplished, and there was a significant difference in the temperature sensitivity between myosin extracted from slow- and fast-twitch fibers. The results from the evaluation of the single fiber in vitro motility assay concludes that this molecular physiological technique offers an exclusive possibility to compare the regulatory influence of various myosin isoforms and thin filament proteins on shortening velocity, at both the cellular and molecular levels. The effects of aging on myosin function were assayed from 82 mouse, rat and human muscle fibers expressing the type I MyHC isoform. The MyLC isoform expression was not affected by aging, in either the rodent or the human fibers. Irrespective of species, an 11-25% aging-related slowing in in vitro motility speed was observed. The 25% decrease in motility speed from 30 months old rats was more than two-fold larger than the 11% slowing observed from myosin of 20-24 months old rats, suggesting an accelerated decline in myosin function with advanced age. In 12 human fibers expressing the IIA MyHC isoform, on the other hand, no difference in motility speed was observed from the young and old myosin. The reduction in type I myosin motility speed indicates that aging-related modifications are taking place in the motor protein, which affects its ability to function optimally. Speeds of actin sliding over myosin extracted from single cells expressing the type I MyHC isoform varied significantly with body size. Motility speed from human myosin was 3-fold slower than from myosin of the ~3400-fold smaller mouse, and approximately 2-fold slower compared with the ~130-fold smaller rat, irrespective of age. The motility speed versus body mass generated a linear and inverse relation when displayed in a double-log plot, suggesting that the observed effects of scaling on actomyosin interactions is related to altered structural and functional properties in the myosin molecule. To explore the effects of glycation as a potential modification underlying the aging-related decline in myosin function, the mechanical properties of the motor protein was tested after incubation with 6 mM glucose for different periods of time. In slow myosin preparations, a reduction by 14 and 17% was observed in actin filament speed after 15- and 20-min incubation respectively, and after 30-min exposure of glucose the motility speed was reduced by 60%. A similar effect on actin gliding speed was observed of myosin isolated from fasttwitch muscle cells. A reversible binding of glucose to the catalytic site of the myosin head is proposed, since addition of hydroxylamine an agent which displaces aldehydes such as glucose from proteins, restored motility speed completely.