Aging is associated with a progressive loss of muscle mass and impairment of muscle contractile functions (i.e., strength, velocity, and power generation). The relevance of those phenomena with age is widely recognized as the life expectancy greatly increases. To date, the underlying mechanisms responsible for muscle weakness and slowing of contractile speed are not clearly identified. Single muscle fiber contractile studies and in vitro motility experiments support the idea that the age-related decline in strength is, in part, because of modifications of myosin (Lowe et al., 2001). Although several studies point to myosin as one of the key players, the reported age-induced slowing of velocity cannot be fully explained by myosin modification or myosin isoform expression. Myosin is composed of two myosin heavy chains (MHCs) and four myosin light chains (MLCs). In rodents, four different types (I, IIA, IIX, IIB) of MHC isoforms are identified. Each MHC consists of two types of MLCs: a pair of essential light chains (ELC) and a pair of regulatory light chains (RLC). In adult skeletal muscle, there are three isoforms of ELC (MLC1f, MLC1s, and MLC3f) and two isoforms of RLC (MLC2f, MLC2s). MHC isoforms are primarily responsible for determining contractile velocity, whereas the ELC isoforms have a modulatory, or fine-tuning, role in the regulation of muscle speed. The contraction speed, as demonstrated by unloaded shortening velocity (Vo), in skeletal muscle increases three to ninefold in the order of MHC type I IIA IIX IIB (Bottinelli et al., 1991, 1994). However, the large variability of Vo within a given MHC fiber type supports the idea that other factors, in addition to MHC, contribute to Vo. Evidence suggests that the variability is, in part, related to the modulatory influence of the ELC isoforms (Sweeney et al., 1988; Lowey et al., 1993a,b; Bottinelli et al., 1994). Using the in vitro motility assay (isolated proteins), the removal of the ELCs from myosin results in a 10-fold decrease in sliding velocity of actin filaments compared with native myosin (Lowey et al., 1993a,b). Likewise, myosin containing only the MLC1f isoform move actin at a significantly slower velocity than myosin containing the MLC3f isoform. Using the permeabilized fiber preparation, MHC type II fibers with a relatively larger amount of MLC3f shorten faster than fibers with a greater amount of MLC1f and Vo is proportional to the relative content of MLC3f (Sweeney et al., 1988; Lowey et al., 1993b; Bottinelli et al., 1994). These results suggest that the relative content of the essential myosin light chains, MLC1f and MLC3f, is an important determinant in the regulation of Vo in MHC type II single fibers.
To date, the aging effect on the relative MLC1f and MLC3f isoform content in skeletal muscle fibers, mainly MHC type II fibers, is unknown. We predict that a decrease in relative MLC3f (concomitant increase in MLC1f content) content with age underlies the slowing of contraction in MHC type II fibers. Therefore, the primary purpose of this study was to use single, permeabilized fibers to determine whether aging decreases the relative MLC3f content (increases the relative MLC1f content) and Vo. The second goal was to restore the loss in Vo with age by increasing MLC3f content via recombinant adenovirus (rAd) gene transfer technology in MHC type II fibers from the semimembranosus (SM) muscle (a muscle composed predominantly of MHC type II fibers). We hypothesize that (i) aging decreases the relative MLC3f content (increases the relative MLC1f content) and Vo in MHC type II SM fibers, and (ii) increasing the MLC3f content by rAd-MLC3f in the SM fibers would restore Vo of individual fibers. We further postulate that rAd-empty vector and MLC3f injection in SM muscle will not result in cellular damage, protein damage, or changes in single fiber diameter and force generation in aged rats.