Fortieth Annual Meeting February 17-21, 1996 Baltimore Convention Center Baltimore, Maryland. Wednesday Symposia and Posters, Part III

1996 Biophysical Journal  
The maximum Ca2+-activated force is also reduced, probably due to myofibrillar disruption. In the present study single fibres were dissected from the mouse flexor brevis muscle and underwent a stretching protocol. The fibres were then examined for ionic and myofibrillar abnormalities using imaging, confocal and electron microscopy. Electron microscopy revealed that there was a considerable variation in the sarcomere lengths of fibres which had undergone the stretching protocol, while in control
more » ... fibres the muscle structure appeared normal. Imaging fluorescent microscopy demonstrated that the distribution of both tetanic and resting [Ca2+]i was uniform along the musclefibres following eccentric contractions. The results show that the single fibre model of eccentric contraction-induced muscle damage is structurally, as well as functionally, akin to the human condition. The results also show that the reduction in tetanic [Ca2+ij occurs uniformly throughout the fibre and is not isolated to specific damaged regions. Calponin is a smooth muscle thin filament-associated protein. To elucidate the physiological role of calponin thisstudy examined the relationship between expression of calponin and postnatal development of contractility in isolated blood vessels. Aortic rings denuded of endothelium from 2, 3, 4, 6, 8, and 10 week-old male Wistar rats were cut to approximately 7, 6, 5, 4, 3 and 2 mm in length, respectively, and the exact tissue mass was used to normalize the data from the different age classes. Each ring was carefully The proposed mechanism of Cal action is via a decrease in the aa2+ off rate with little or no effect on the Ca 2 on rate (Wahr et aL, 1993 BiophysJ. 64:A24; Johnson et al., 1994, JBC 269:8919). If this mechanism is correct Cal should affect force generation by muscle fibers at low (Ca21 but not at maximal Ca2+ activation. We tested this hypothesis by measuring the stead2ystate isometric force (P) and the rate of force development (k )in Ca Ca activated, skinned fibers from rabbit psoas. At maximal Ca2+ activation (pCa 4.0) [Cal] 3 u M had no discernible effect on either P0 or ki, At 100 M Cal both Po and kir were reduced by 20-30%, an effect that was at least partially reversible. In contrast, P and kr, were both increased at submaximnal Ca2+ activations in the presence of Cal (relative to control values). At 30 iM Cal the pCa-force curve was shifted to the left by approximately 0.2 pCa units. Additionally, 30 tiM Cal increased kr, by 2-3 fold at 50% P,. These effects at submaximal [Ca 21 were not readily reversible. These results support the hypothesis that the Ca2d binding propertiea of TnC are important modulators of the rate of force development in skeletal muscle (Chase et al., Decoration of thin filaments in "ghost fibers" with NEM-modified myosin subfragment-I and phosphorylated smooth muscle heavy meromyosin produces structural changes in actin which is typical for the formation of 'strong binding" between actin and the myosin head. The effects of actingbinding proteins calponin and 38 kDa fragment of caldesmon on these structural changes was investigated using polarized fluorometry.The F-actin in the myosin-free "ghost fibers" was labeled with fluorescein-maleimide and TRITC-phalloidin. Both actin-binding regulatory proteins inhibited the conformational changes in actin, that are compatible with the formation of "strong binding" between actin and the myosin head. Tropomyosin reduced the effect produced by calponin, but increased the conformational change caused by the 38 kDa caldesmon.The in vitro motility assay showed that these regulatory proteins inhibit motility of actin filament over immobilized surface of skeletal muscle myosin heads. It is thus suggested that the inhibition of the formation of "strong-binding" is an important factor in the mechanism for the regulation of smooth muscle contraction. Supp. by grants from RBRF& NIH. Muscle from the giant barnacle (Balanus nubilus) has been shown to contain two isoforms of Troponin C, BTnC, and BTnC, (Ashley et al., 1991). The amino acid sequences of both isoforms have been determined and the Ca" binding properties of BTnC2 have been characterized (Collins et al., 1991). BTnC2 binds two moles of Ca'-/mole and both sites appear to be Cal'-specific. Analysis of the amino acid sequence suggests that only sites II and IV bind Ca". In order to learn more about the possible structure ofthis protein, we built a three dimensional model of BTnC, using a computer program (Homology: Biosym, Inc.) that constructs an unk-nown protein structure from proteins with homologous sequence and/or conserved structural regions. After constructing our protein we used a unique iterative minimization and dynamics simulation method which prevented loss of structure due to excessive intramolecular stress. Our structure was dumbbell shaped containing N-and C-terminal halves. The N-terminus is shorter than the turkey/chick-en crystal structures, however it is also comprised of a-helix. Sites I through IV are all composed of helixloop-helix structures however site III is altered from a "classic" EF hand motif. Although the D/E link-er and region IV have sequences that differ significantlv from analogous regions of vertebrates, they appear to be similar in structure. (Supported bv We investigated the role of phosphorylation of the myosin regulatory light chains (RLCs) in the regulation of skeletal muscle contraction. The RLCs were phosphorylated in skinned skeletal muscle fibers with Ca2+-calmodulin activated myosin light chain kinase (MLCK). This treatment resulted in an -10-20% increase in steady state force development compared to untreated fibers. The Ca2' sensitivity of force development was also affected by RLCs phosphorylation. Consistent with Metzger et at., J. Gen. Physiol. 93: 855-883 (1989) and Sweeney et aL, Am. J. Physiol. 264: C1085-C1095 (1993), the force -pCa relationship was shifted towards lower concentrations of Ca2e compared to non-phosphorylated fibers. The same results were obtained when steady state force measurements were performed on skinned fibers extracted to remove endogenous RLCs (Szczesna et al., Biophys. J. 68: A168, 1995) and reconstituted with either phosphorylated RLCs (+P-RLCs) or nonphosphorylated RLCs that were subsequently phosphorylated with MLCK. In both cases we observed an -30% increase in maximal force and an increase in the Ca2, sensitivity of force development of &pCaso-+0.13 of the +P-RLCs-reconstituted fibers compared to fibers reconstituted with non-phosphorylated RLCs. These results suggest that although skeletal muscle is primarily regulated by the troponintropomyosin complex, phosphorylation of the regulatory light chains of myosin may play an important modulatory role in vertebrate striated muscle contraction. The changes in actin conformation induced by the binding of phosphorylated and dephosphorylated heavy meromyosin (HMM) were determined by measuring the polarized fluorescence of rhodamine-phalloidin complex attached to F-actin. Phosphorylated HMM in the presence of Ca2" induced the conformational changes on actin typical for the "on" state of actin monomers in thin filaments, a specific change caused by "strong-binding" of myosin heads. This effect was markedly inhibited in the absence of Ca2' and when HMM light chain was dephosphorylated. Therefore, it is suggested that both Ca2" and phosphorylation of myosin regulatory light chains switch myosin heads from "weak binding" to a "strong-binding" conformation and the binding of myosin heads switches actin monomers from "off" to "on" state. In skeletal and cardiac muscle, contraction and relaxation is regulated by the binding of calcium to troponin C (TnC), the calcium binding subunit of the troponin complex. The conformatonal change induced in TnC by calcium is transmiaed through the remaining two subunits, troponin T (Tnl) and troponin I (Tnl), eventually reaching actin and myosin, allowing contraction to occur. Recent studies (Potter, et al., J. Biol. Chem. M.: 2557, (1995) has suggested an important role for TnT in this process. In order to understand these conformational changes, we have chosen to study the structure of TnT. Initial experiments have been performed with TnT isolated from rabbit cardiac and skeletal muscle. Quasi-elastic light scattering experiments indicate that TnT is an extremely elongated molecule, with cardiac TnT being approximately fifty angstroms longer than skeletal TnT. Circular dichroism studies also indicate differences in structurc between these two proteins, with TnT from cardiac muscle being approximately 20% more a-helical than skeletal muscle TnT. This work is being continued on recombinant TnT-a, an individual isoform from rabbit skeletal muscle. From this clone, we have also constructed and expressed two deletion mutants, Ti (residues 1-149) and T2 (residues 150-250). From the biophysical characterizations of both the intact protein and the deletion mutants, we intend to determine more precisely the secondary and tertiary structure of TnT as a prelude to a full description of the overall structure of the troponin complex.
doi:10.1016/s0006-3495(96)79672-8 fatcat:ohs2vrzsxvbppaarefqlgaya5u