Increase of skeletal muscle relaxation speed by direct injection of parvalbumin cDNA.

Parvalbumin (PV) is a high affinity Ca(2+)-binding protein found at high concentration in fast-contracting/relaxing skeletal muscle fibers of vertebrates. It has been proposed that PV acts in the process of muscle relaxation by facilitating Ca2+ transport from the myofibrils to the sarcoplasmic reticulum. However, on the basis of metal-binding kinetics of PV in vitro, this hypothesis has been challenged. To investigate the function of PV in skeletal muscle fibers, direct gene transfer was applied in normal and regenerating rat soleus muscles which do not synthesize detectable amounts of PV. Two weeks after in vivo transfection with PV cDNA, considerable levels of PV mRNA and protein were detected in normal muscle, and even higher amounts were detected in regenerating muscle. Twitch half-relaxation time was significantly shortened in a dose-dependent way in transfected muscles, while contraction time remained unaltered. The observed shortening of half-relaxation time is due to PV and its ability to bind Ca2+, because a mutant protein lacking Ca(2+)-binding capacity did not promote any change in physiology. These results directly demonstrate the physiological function of PV as a relaxing factor in mammalian skeletal muscle.

Contraction stimulates translocation of glucose transporter GLUT4 in skeletal muscle through a mechanism distinct from that of insulin.

The acute effects of contraction and insulin on the glucose transport and GLUT4 glucose transporter translocation were investigated in rat soleus muscles by using a 3-O-methylglucose transport assay and the sensitive exofacial labeling technique with the impermeant photoaffinity reagent 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannose-4-y loxy)-2- propylamine (ATB-BMPA), respectively. Addition of wortmannin, which inhibits phosphatidylinositol 3-kinase, reduced insulin-stimulated glucose transport (8.8 +/- 0.5 mumol per ml per h vs. 1.4 +/- 0.1 mumol per ml per h) and GLUT4 translocation [2.79 +/- 0.20 pmol/g (wet muscle weight) vs. 0.49 +/- 0.05 pmol/g (wet muscle weight)]. In contrast, even at a high concentration (1 microM), wortmannin had no effect on contraction-mediated glucose uptake (4.4 +/- 0.1 mumol per ml per h vs. 4.1 +/- 0.2 mumol per ml per h) and GLUT4 cell surface content [1.75 +/- 0.16 pmol/g (wet muscle weight) vs. 1.52 +/- 0.16 pmol/g (wet muscle weight)]. Contraction-mediated translocation of the GLUT4 transporters to the cell surface was closely correlated with the glucose transport activity and could account fully for the increment in glucose uptake after contraction. The combined effects of contraction and maximal insulin stimulation were greater than either stimulation alone on glucose transport activity (11.5 +/- 0.4 mumol per ml per h vs. 5.6 +/- 0.2 mumol per ml per h and 9.0 +/- 0.2 mumol per ml per h) and on GLUT4 translocation [4.10 +/- 0.20 pmol/g (wet muscle weight) vs. 1.75 +/- 0.25 pmol/g (wet muscle weight) and 3.15 +/- 0.18 pmol/g (wet muscle weight)]. The results provide evidence that contraction stimulates translocation of GLUT4 in skeletal muscle through a mechanism distinct from that of insulin.

Rates of ubiquitin conjugation increase when muscles atrophy, largely through activation of the N-end rule pathway

The rapid loss of muscle mass that accompanies many disease states, such as cancer or sepsis, is primarily a result of increased protein breakdown in muscle, and several observations have suggested an activation of the ubiquitin–proteasome system. Accordingly, in extracts of atrophying muscles from tumor-bearing or septic rats, rates of 125I-ubiquitin conjugation to endogenous proteins were found to be higher than in control extracts. On the other hand, in extracts of muscles from hypothyroid rats, where overall proteolysis is reduced below normal, the conjugation of 125I-ubiquitin to soluble proteins decreased by 50%, and treatment with triiodothyronine (T3) restored ubiquitination to control levels. Surprisingly, the N-end rule pathway, which selectively degrades proteins with basic or large hydrophobic N-terminal residues, was found to be responsible for most of these changes in ubiquitin conjugation. Competitive inhibitors of this pathway that specifically block the ubiquitin ligase, E3α, suppressed most of the increased ubiquitin conjugation in the muscle extracts from tumor-bearing and septic rats. These inhibitors also suppressed ubiquitination in normal extracts toward levels in hypothyroid extracts, which showed little E3α-dependent ubiquitination. Thus, the inhibitors eliminated most of the differences in ubiquitination under these different pathological conditions. Moreover, 125I-lysozyme, a model N-end rule substrate, was ubiquitinated more rapidly in extracts from tumor-bearing and septic rats, and more slowly in those from hypothyroid rats, than in controls. Thus, the rate of ubiquitin conjugation increases in atrophying muscles, and these hormone- and cytokine-dependent responses are in large part due to activation of the N-end rule pathway.

The whistle and the rattle: the design of sound producing muscles.

Vertebrate sound producing muscles often operate at frequencies exceeding 100 Hz, making them the fastest vertebrate muscles. Like other vertebrate muscle, these sonic muscles are "synchronous," necessitating that calcium be released and resequestered by the sarcoplasmic reticulum during each contraction cycle. Thus to operate at such high frequencies, vertebrate sonic muscles require extreme adaptations. We have found that to generate the "boatwhistle" mating call (approximately 200 Hz), the swimbladder muscle fibers of toadfish have evolved (i) a large and very fast calcium transient, (ii) a fast crossbridge detachment rate, and (iii) probably a fast kinetic off-rate of Ca2+ from troponin. The fibers of the shaker muscle of rattlesnakes have independently evolved similar traits, permitting tail rattling at approximately 90 Hz.

Functional protection of dystrophic mouse (mdx) muscles after adenovirus-mediated transfer of a dystrophin minigene.

Fast skeletal muscles of mdx (X chromosome-linked muscular dystrophy) mice were injected after birth with a recombinant adenovirus containing a minidys- trophin gene, a 6.3-kbp cDNA coding for the N- and C-terminal ends of dystrophin. Adult muscles were challenged by forced lengthening during tetanic contractions. Stretch-induced mechanical and histological damages were much reduced in injected muscles, in direct proportion of the Miniber of fibers expressing minidystrophin. Damaged fibers were preferentially found among minidystrophin-negative regions. Minidystrostrophin confers an important functional and structural protection of limb muscles against high mechanical stress, even after a partial somatic gene transfer.