No abstract

In previous efforts to characterize sarcoplasmic reticulum function in human muscles, it has not been possible to distinguish the relative contributions of fast-twitch and slow-twitch fibers . In this study, we have used light scattering and 45Ca to monitor Ca accumulation by the sarcoplasmic reticulum of isolated, chemically skinned human muscle fibers in the presence and absence of oxalate . Oxalate (5 mM) increased the capacity for Ca accumulation by a factor of 35 and made it possible to assess both rate of Ca uptake and relative sarcoplasmic reticulum volume in individual fibers . At a fixed ionized Ca concentration, the rate and maximal capacity (an index of sarcoplasmic reticulum volume) both varied over a wide range, but fibers fell into two distinct groups (fast and slow) . Between the two groups, there was a 2-to 2.5-fold difference in oxalate-supported Ca uptake rates, but no difference in average sarcoplasmic reticulum volumes . Intrinsic differences in sarcoplasmic reticulum function (V., Ko.s, and n) were sought to account for the distinction between fast and slow groups . In both groups, rate of Ca accumulation increased sigmoidally as [Ca"] was increased from 0.1 to 1 jiM . Apparent affinities for Ca" (Ko.s) were similar in the two groups, but slow fibers had a lower V.. and larger n values . Slow fibers also differed from fast fibers in responding with enhanced Ca uptake rates upon addition of cyclic AMP (10-s M, alone or with protein kinase) . Acceleration by cyclic AMP was adequate to account for adrenaline-induced increases in relaxation rates previously observed in human muscles containing mixtures of fast-twitch and slow-twitch fibers .

The contraction of skeletal muscle is regulated by calcium binding to troponin C (TnC). TnC consists of two spatially independent domains, each of which contains two metal ion binding sites. Calcium binding to the regulatory sites of the N-terminal domain triggers muscle contraction by a series of conformational changes. Site-directed mutagenesis offers a means of elucidating the links in this signal path between TnC and actin-myosin crossbridges. Such mapping is possible if the mutants shift the equilibrium between 'on' and 'off' states of the regulatory complex while maintaining the coupling between calcium binding and tension development. Candidate amino-acid residues for yielding this information would be in positions remote from the calcium-binding sites and from the site of development of tension. Analysis of the crystal structure of TnC and of the model of the calcium-activated molecule has enabled us to identify two such residues: Glu 57 and Glu 88. In separate experiments we have replaced each of these residues by lysines. The resulting reduction in calcium affinity indicates that these residues have a long-range effect on calcium binding. This result may reflect the formation of a salt bridge between positions 57 and 88 that is not present in the native molecule. Moreover, the level of tension recovery when the mutants are incorporated into muscle suggests that the interaction between TnC and other muscle components has also been altered. Thus, these residues may participate in the contraction signal transmission.

Nitric oxide (NO*) is synthesized in skeletal muscle and its production increases during contractile activity. Although myosin is the most abundant protein in muscle, it is not known whether myosin is a target of NO* or NO* derivatives. In the present study, we have shown that exercise increases protein S-nitrosylation in muscle, and, among contractile proteins, myosin is the principal target of exogenous SNOs (S-nitrosothiols) in both skinned skeletal muscle fibres and differentiated myotubes. The reaction of isolated myosin with S-nitrosoglutathione results in S-nitrosylation at multiple cysteine thiols and produces two populations of protein-bound SNOs with different stabilities. The less-stable population inhibits the physiological ATPase activity, without affecting the affinity of myosin for actin. However, myosin is neither inhibited nor S-nitrosylated by the NO* donor diethylamine NONOate, indicating a requirement for transnitrosylation between low-mass SNO and myosin cysteine thiols rather than a direct reaction of myosin with NO* or its auto-oxidation products. Interestingly, alkylation of the most reactive thiols of myosin by N-ethylmaleimide does not inhibit formation of a stable population of protein-SNOs, suggesting that these sites are located in less accessible regions of the protein than those that affect activity. The present study reveals a new link between exercise and S-nitrosylation of skeletal muscle contractile proteins that may be important under (patho)physiological conditions.