PK1 from Drosophila obscurin is an inactive pseudokinase with scaffolding properties.

Obscurins are large filamentous proteins with crucial roles in the assembly, stability and regulation of muscle. Characteristic of these proteins is a tandem of two C-terminal kinase domains, PK1 and PK2, that are separated by a long intrinsically disordered sequence. The significance of this conserved domain arrangement is unknown. Our study of PK1 from Drosophila obscurin shows that this is a pseudokinase with features typical of the CAM-kinase family, but which carries a minimalistic regulatory tail that no longer binds calmodulin or has mechanosensory properties typical of other sarcomeric kinases. PK1 binds ATP with high affinity, but in the absence of magnesium and lacks detectable phosphotransfer activity. It also has a highly diverged active site, strictly conserved across arthropods, that might have evolved to accommodate an unconventional binder. We find that PK1 interacts with PK2, suggesting a functional relation to the latter. These findings lead us to speculate that PK1/PK2 form a pseudokinase/kinase dual system, where PK1 might act as an allosteric regulator of PK2 and where mechanosensing properties, akin to those described for regulatory tails in titin-like kinases, might now reside on the unstructured interkinase segment. We propose that the PK1-interkinase-PK2 region constitutes an integrated functional unit in obscurin proteins.

SarConfoCal: simultaneous sarcomere length and cytoplasmic calcium measurements for laser scanning confocal microscopy images.

Summary: Simultaneous recordings of myocytes contractility and their cytoplasmic calcium concentration allow powerful studies, particularly on heart failure and other cardiac dysfunctions. Such studies require dedicated and expensive experimental devices that are difficult to use. Thus we propose SarConfoCal, the first and only software to simultaneously analyse both cytoplasmic calcium variations (from fluorescence signal) and myocytes contractility (from sarcomere length measurement) on laser scanning confocal microscopy images. SarConfoCal is easy to set up and use, especially by people without programming skills. Availability and implementation: The software is freely distributed under the GNU General Public License. Download and setup instructions are available at http://pccv.univ-tours.fr/ImageJ/SarConfoCal . It is provided as a toolset for ImageJ (the open-source program for image analysis provided by the National Institutes of Health). SarConfoCal has been tested under Windows, Mac and Linux operating systems. Contact: come.pasqualin@univ-tours.fr. Supplementary information: Supplementary data are available at Bioinformatics online.

The influence of peri-conception and first trimester dietary restriction of protein in cattle on meat quality traits of entire male progeny.

Primiparous Santa Gertrudis heifers were used to evaluate the effects of gestational dietary protein content on meat quality traits of 20month old bull progeny (n=40). At -60d before AI, heifers were randomly allocated to HIGH or LOW protein diet (HPERI and LPERI). From 24dpc, half of each treatment group changed to an alternative post-conception HIGH or LOW protein diet (HPOST and LPOST). LPERI and LPOST diets resulted in higher shear force of the semitendinosus muscle than HPERI (P=0.053) and HPOST (P=0.003), respectively. Heat-soluble collagen in the semitendinosus muscle was lower (P=0.019) for LPERI than HPERI. Collagen and tenderness of the longissimus muscle were not affected by dam nutrition (P>0.05). Color, pH, sarcomere length, cooking loss, compression values, desmin and troponin-T degradation, fiber type, intramuscular fat and polyunsaturated fatty acid content were not affected by dam nutrition during the peri-conception and first trimester gestational period (P>0.05).

Electron-microscopical localization of gelsolin in various crustacean muscles.

Gelsolin was localized by immunoelectron microscopy in fast and slow cross-striated muscles of the lobster Homarus americanus. When ultrathin sections of the muscles were labelled with anti-gelsolin and a gold-conjugated second antibody, 90% of all gold particles in the myoplasm were detected on myofibrils, preferentially in the I-band and AI-region of the sarcomeres. Both the region of the H-zone (lacking thin filaments) and the Z-disc contained no or little gold label. Under physiological conditions, a close association of gelsolin with the thin filaments was observed for both muscle types. The preferential localization of particles in the I- and AI-region indicated that gelsolin was distributed randomly over the whole length of the thin filaments. Preincubation of muscle strips with Ringer solution containing 0.5 mM EGTA resulted in a significantly different distribution pattern; gold particles were now localized preferentially in the cell periphery close to the sarcolemma, with significantly decreased abundance in the centre of the cell. Compared with the muscle under physiological conditions, the number of gold particles over sarcomeric structures was significantly reduced. Thus, binding of gelsolin to the thin filaments is apparently reversible in vivo and depends on the presence of calcium ions. We assume a functional role for gelsolin in the actin turnover processes in invertebrate muscle systems.

Force, sarcomere shortening velocity and ATPase activity.

UNLABELLED: We have tested the hypothesis that the transition rate (G) of the cardiac XB from the strong force generating state to the weak state is a linear function V of the sarcomere (VSL); furthermore, we tested whether the ATPase rate of the two isoforms of myosin can be held responsible for the difference between V0 of rat cardiac trabeculae containing V1 isomyosin versus those containing V3 isomyosin. METHODS: V1 isomyosin was induced by thyroid hormone treatment of the rats for 2 weeks, V3 isomyosin by PTU treatment for 1 month. Force was measured with a strain gauge in trabeculae from the rat right ventricle in K-H solution ([Ca]o=1.5 mM, 25 degrees C). Sarcomere length (SL) was measured with laser diffraction techniques. Twitch force at constant SL, and the force response to shortening at constant VSL (0-8 microm/s; deltaSL 50-100 nm) were measured at varied time during the twitch. RESULTS: The force response to shortening consisted of a fast initial exponential decline (tau = 2 ms) followed by a slow decrease of F. The instantaneous difference (deltaF) between isometric force (FM) and the declining force depended on shortening duration (deltat), VSL and instantaneous FM: deltaF = G1 x FM x deltat x VSL x (1-VSL/VMAX), where VMAX is the unloaded VSL and G1 was 6.15 +/- 2.12 microm(-1) (mean +/- s.d.; n=6). deltaF/FM was independent of the time onset of shortening. G1 of V1 and V3 trabeculae did not differ. V0 of V1 and V3 trabeculae differed 2-2.5 fold, as did both the ATPase rate and the velocity of actin sliding in a motility assay of the myosin purified from V1 or V3 hearts. The temperature dependence of the ATPase rate (Q10: 4.03 and 4.33, respectively; n.s.) was similar to that of V0 that has previously been reported for predominantly V1 trabeculae. Cross-linking of actin to myosin with the short chain cross linker EDC increased the ATPase rate of the two isomyosins (200-fold and 600-fold respectively) to exactly the same final level and reduced their Q10 by 50%. CONCLUSION: The linear interrelation between deltaF and VSL is consistent with feedback, whereby XB kinetics depends on VSL. This feedback provides an integrated description of cardiac muscle mechanics and energetics. The results, also, suggests that it is unlikely that the hydrolytic domain of the cross bridge determines V0 and warrant ongoing experiments to investigate the role of the actin binding domain of the XB in cardiac sarcomere kinetics. In order to further investigate the role of the actin binding domain, we have expressed chimeric cardiac myosin, co-assembled with MLC, by mutual substitution of actin binding loop on alpha MHC and beta MHC.

Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly.

Vertebrate-striated muscle is assumed to owe its remarkable order to the molecular ruler functions of the giant modular signaling proteins, titin and nebulin. It was believed that these two proteins represented unique results of protein evolution in vertebrate muscle. In this paper we report the identification of a third giant protein from vertebrate muscle, obscurin, encoded on chromosome 1q42. Obscurin is approximately 800 kD and is expressed specifically in skeletal and cardiac muscle. The complete cDNA sequence of obscurin reveals a modular architecture, consisting of >67 intracellular immunoglobulin (Ig)- or fibronectin-3-like domains with multiple splice variants. A large region of obscurin shows a modular architecture of tandem Ig domains reminiscent of the elastic region of titin. The COOH-terminal region of obscurin interacts via two specific Ig-like domains with the NH(2)-terminal Z-disk region of titin. Both proteins coassemble during myofibrillogenesis. During the progression of myofibrillogenesis, all obscurin epitopes become detectable at the M band. The presence of a calmodulin-binding IQ motif, and a Rho guanine nucleotide exchange factor domain in the COOH-terminal region suggest that obscurin is involved in Ca(2+)/calmodulin, as well as G protein-coupled signal transduction in the sarcomere.

Point mutations in human beta cardiac myosin heavy chain have differential effects on sarcomeric structure and assembly: an ATP binding site change disrupts both thick and thin filaments, whereas hypertrophic cardiomyopathy mutations display normal assembly.

Hypertrophic cardiomyopathy is a human heart disease characterized by increased ventricular mass, focal areas of fibrosis, myocyte, and myofibrillar disorganization. This genetically dominant disease can be caused by mutations in any one of several contractile proteins, including beta cardiac myosin heavy chain (beta MHC). To determine whether point mutations in human beta MHC have direct effects on interfering with filament assembly and sarcomeric structure, full-length wild-type and mutant human beta MHC cDNAs were cloned and expressed in primary cultures of neonatal rat ventricular cardiomyocytes (NRC) under conditions that promote myofibrillogenesis. A lysine to arginine change at amino acid 184 in the consensus ATP binding sequence of human beta MHC resulted in abnormal subcellular localization and disrupted both thick and thin filament structure in transfected NRC. Diffuse beta MHC K184R protein appeared to colocalize with actin throughout the myocyte, suggesting a tight interaction of these two proteins. Human beta MHC with S472V mutation assembled normally into thick filaments and did not affect sarcomeric structure. Two mutant myosins previously described as causing human hypertrophic cardiomyopathy, R249Q and R403Q, were competent to assemble into thick filaments producing myofibrils with well defined I bands, A bands, and H zones. Coexpression and detection of wild-type beta MHC and either R249Q or R403Q proteins in the same myocyte showed these proteins are equally able to assemble into the sarcomere and provided no discernible differences in subcellular localization. Thus, human beta MHC R249Q and R403Q mutant proteins were readily incorporated into NRC sarcomeres and did not disrupt myofilament formation. This study indicates that the phenotype of myofibrillar disarray seen in HCM patients which harbor either of these two mutations may not be directly due to the failure of the mutant myosin heavy chain protein to assemble and form normal sarcomeres, but may rather be a secondary effect possibly resulting from the chronic stress of decreased beta MHC function.