Influence of locomotor training on the structure and myosin heavy chains of the denervated rat soleus muscle.

OBJECTIVE: Mechanism of denervation atrophy remains poorly understood. In particular, the question about irreversibility of the late atrophy is still open. Therefore, in the present study, we investigated whether and how a passive movement can affect a progress of atrophy in rat soleus muscle. To address this issue, a locomotor training on a treadmill was applied to rats with their right hindlimb muscles denervated. METHODS: The hindlimb muscles were denervated by cutting the sciatic nerve. Starting either 7 days or 1 month after the surgery, the animals were trained on a treadmill. Two months after denervation, the soleus muscle was investigated using light and electron microscopy and biochemical methods. Control soleus muscles were obtained from non-trained animals: the untreated and the 2-month denervated. RESULTS: Locomotor training caused slight increase in denervated rat soleus muscle weight and significant increase in its fiber diameter. The training positively affected some of the factors that were believed to be the reasons of atrophy irreversibility, because of significant increase in the number of capillary blood vessels and muscle fiber nuclei with the concomitant decrease in the number of severely damaged muscle fibers and amount of collagen. Morphology of the contractile apparatus was also improved as more regular organization of sarcomeres and the hexagonal arrangement of myosin filaments was evident. Moreover, the amount of myosin heavy chains (MHC) significantly increased after training. The effects were more evident in the animals with longer training. CONCLUSION: Passive movement seems to attenuate some of the pathologic processes within the denervated muscle.

Ionic interaction of myosin loop 2 with residues located beyond the N-terminal part of actin probed by chemical cross-linking.

To probe ionic contacts of skeletal muscle myosin with negatively charged residues located beyond the N-terminal part of actin, myosin subfragment 1 (S1) and actin split by ECP32 protease (ECP-actin) were cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). We have found that unmodified S1 can be cross-linked not only to the N-terminal part, but also to the C-terminal 36 kDa fragment of ECP-actin. Subsequent experiments performed on S1 cleaved by elastase or trypsin indicate that the cross-linking site in S1 is located within loop 2. This site is composed of Lys-636 and Lys-637 and can interact with negatively charged residues of the 36 kDa actin fragment, most probably with Glu-99 and Glu-100. Cross-links are formed both in the absence and presence of MgATP.P(i) analog, although the addition of nucleotide decreases the efficiency of the cross-linking reaction.

Effect of nucleotide on interaction of the 567-578 segment of myosin heavy chain with actin.

To probe the effect of nucleotide on the formation of ionic contacts between actin and the 567-578 residue loop of the heavy chain of rabbit skeletal muscle myosin subfragment 1 (S1), the complexes between F-actin and proteolytic derivatives of S1 were submitted to chemical cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. We have shown that in the absence of nucleotide both 45 kDa and 5 kDa tryptic derivatives of the central 50 kDa heavy chain fragment of S1 can be cross-linked to actin, whereas in the presence of MgADP.AlF4, only the 5 kDa fragment is involved in cross-linking reaction. By the identification of the N-terminal sequence of the 5-kDa fragment, we have found that trypsin splits the 50 kDa heavy chain fragment between Lys-572 and Gly-573, the residues located within the 567-578 loop. Using S1 preparations cleaved with elastase, we could show that the residue of 567-578 loop that can be cross-linked to actin in the presence of MgADP.AlF4 is Lys-574. The observed nucleotide-dependent changes of the actin-subfragment 1 interface indicate that the 567-578 residue loop of skeletal muscle myosin participates in the communication between the nucleotide and actin binding sites.

Inhibitory effect of ATP analogs and actin on the modification of myosin subfragment 1 with 9-anthroylnitrile.

The fluorescent probe, 9-anthroylnitrile (ANN), can selectively attach to Ser-180 at the ATP-binding site of subfragment 1 (S1) of skeletal muscle myosin [J. Biol. Chem. 278 (2003) 31891]. We have found that MgATP, MgATPgammaS, MgADP.AlF(4) or MgPP(i), but not MgADP, inhibit the incorporation of ANN into S1. The inhibitory effect of the nucleotide gamma-phosphate group (or its analog) on the modification of S1 with ANN can be explained by the contribution of Ser-180 to the binding of the nucleotide gamma-phosphate at the active site of S1. We have also observed that the incorporation of ANN into S1.MgADP complex is inhibited by actin. These experimental data strongly support the existence of nucleotide-promoted conformational changes revealed by crystal structures of S1 complexes with various nucleotide analogs. They also convincingly show an effect of actin on the environment of Ser-180 at the nucleotide binding site of S1.

Changes at the interface of the N- and C-terminal parts of the heavy chain of myosin subfragment 1.

It has been previously shown that in the M-MgADP-P(i) state, where the myosin head adopts a pre-power stroke conformation, treatment of trypsin-split subfragment 1 of skeletal muscle myosin with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) results in cross-linking of the C-terminal fragment of the heavy chain of S1 -- most probably its converter region -- to the N-terminal S1 heavy-chain fragment, generating a product of 44 kDa [Biochim. Biophys. Acta 1481 (2000) 55]. The results described here show that this product is neither generated in the absence of nucleotide nor in the presence of MgADP or MgPP(i). The 44 kDa cross-linking product can be formed when S1 treated with EDC is complexed with MgADP-AlF(4) or MgADP-V(i) (MgADP-P(i) analogs) and with MgADP-BeF(x), MgATP gamma S or MgAMPPNP (MgATP analogs). The results suggest structural differences between MgATP- or MgADP-P(i)-bound S1, and MgADP-bound or nucleotide-free S1, in spatially close regions of their N- and C-terminal heavy-chain fragments.