Abstract-Myosin binding protein C (MyBP-C) is one of the major sarcomeric proteins involved in the pathophysiology of familial hypertrophic cardiomyopathy (FHC). The cardiac isoform is tris-phosphorylated by cAMP-dependent protein kinase (cAPK) on ␤-adrenergic stimulation at a conserved N-terminal domain (MyBP-C motif), suggesting a role in regulating positive inotropy mediated by cAPK. Recent data show that the MyBP-C motif binds to a conserved segment of sarcomeric myosin S2 in a phosphorylation-regulated way. Given that most MyBP-C mutations that cause FHC are predicted to result in N-terminal fragments of the protein, we investigated the specific effects of the MyBP-C motif on contractility and its modulation by cAPK phosphorylation. The diffusion of proteins into skinned fibers allows the investigation of effects of defined molecular regions of MyBP-C, because the endogenous MyBP-C is associated with few myosin heads. Furthermore, the effect of phosphorylation of cardiac MyBP-C can be studied in a defined unphosphorylated background in skeletal muscle fibers only. Triton skinned fibers were tested for maximal isometric force, Ca 2ϩ /force relation, rigor force, and stiffness in the absence and presence of the recombinant cardiac MyBP-C motif. The presence of unphosphorylated MyBP-C motif resulted in a significant (1) depression of Ca 2ϩ -activated maximal force with no effect on dynamic stiffness, (2) increase of the Ca 2ϩ sensitivity of active force (leftward shift of the Ca 2ϩ /force relation), (3) increase of maximal rigor force, and (4) an acceleration of rigor force and rigor stiffness development. Tris-phosphorylation of the MyBP-C motif by cAPK abolished these effects. This is the first demonstration that the S2 binding domain of MyBP-C is a modulator of contractility. The anchorage of the MyBP-C motif to the myosin filament is not needed for the observed effects, arguing that the mechanism of MyBP-C regulation is at least partly independent of a "tether," in agreement with a modulation of the head-tail mobility. Soluble fragments occurring in FHC, lacking the spatial specificity, might therefore lead to altered contraction regulation without affecting sarcomere structure directly. (Circ Res. 2000;86:51-58.)

Using laser scanning confocal microscopy, we show for the first time elementary Ca2+ release events (ECRE) from the sarcoplasmic reticulum in chemically and mechanically skinned fibres from adult mammalian muscle and compare them with ECRE from amphibian skinned fibres. Hundreds of spontaneously occurring events could be measured from individual single skinned mammalian fibres. In addition to spark‐like events, we found ember‐like events, i.e. long‐lasting events of steady amplitude. These two different fundamental release types in mammalian muscle could occur in combination at the same location. The two peaks of the frequency of occurrence for ECRE of mammalian skeletal muscle coincided with the expected locations of the transverse tubular system within the sarcomere, suggesting that ECRE mainly originate at triadic junctions. ECRE in adult mammalian muscle could also be identified at the onset of the global Ca2+ release evoked by membrane depolarisation in mechanically skinned fibres. In addition, the frequency of ECRE was significantly increased by application of 0.5 mm caffeine and reduced by application of 2 mm tetracaine. We conclude that the excitation‐contraction coupling process in adult mammalian muscle involves the activation of both spark‐ and ember‐like elementary Ca2+ release events.

We use second harmonic generation (SHG) imaging to study and quantify a strong intrinsic SHG signal in skeletal muscle fiber preparations and single isolated myofibrils. The intrinsic signal follows the striation pattern of the muscle cells and is positioned at the sarcomeric location of the myosin filaments. Interestingly, the signal is enhanced at the region where the myosin heads are located on the myosin filaments. As the intrinsic signal reflects the subcellular structure in an accurate way, SHG can be used for noninvasive high resolution structural imaging without exogenous labels in living muscle cells. This may be very important for detecting changes in myofibrillar organization occurring under pathophysiological conditions, e.g., in cardiac and skeletal myopathies. Due to the strong dependency of SHG on orientation and symmetries of the tissue, it may allow the study of dynamic interactions between the contractile proteins actin and myosin during force production and muscle shortening. Furthermore, SHG imaging can be combined with other nonlinear microscopical techniques, such as laser scanning multiphoton fluorescence microscopy, to simultaneously measure other dynamic cellular processes, representing a complementary method and extending the range of nonlinear microscopical methods.

The recent observation that central refractive development might be controlled by the refractive errors in the periphery, also in primates, revived the interest in the peripheral optics of the eye. We optimized an eccentric photorefractor to measure the peripheral refractive error in the vertical pupil meridian over the horizontal visual field (from -45 degrees to 45 degrees ), with and without myopic spectacle correction. Furthermore, a newly designed radial refractive gradient lens (RRG lens) that induces increasing myopia in all radial directions from the center was tested. We found that for the geometry of our measurement setup conventional spectacles induced significant relative hyperopia in the periphery, although its magnitude varied greatly among different spectacle designs and subjects. In contrast, the newly designed RRG lens induced relative peripheral myopia. These results are of interest to analyze the effect that different optical corrections might have on the emmetropization process.

S100A1, a Ca2؉ -binding protein of the EF-hand type, is most highly expressed in striated muscle and has previously been shown to interact with the skeletal muscle sarcoplasmic reticulum (SR) Ca 2؉ release channel/ryanodine receptor (RyR1) isoform. However, it was unclear whether S100A1/RyR1 interaction could modulate SR Ca 2؉ handling and contractile properties in skeletal muscle fibers. Since S100A1 protein is differentially expressed in fast-and slow-twitch skeletal muscle, we used saponin-skinned murine Musculus extensor digitorum longus (EDL) and Musculus soleus (Soleus) fibers to assess the impact of S100A1 protein on SR Ca 2؉ release and isometric twitch force in functionally intact permeabilized muscle fibers. S100A1 equally enhanced caffeine-induced SR Ca 2؉ release and Ca 2؉ -induced isometric force transients in both muscle preparations in a dose-dependent manner. Introducing a synthetic S100A1 peptide model (devoid of EF-hand Ca 2؉ -binding sites) allowed identification of the S100A1 C terminus (amino acids 75-94) and hinge region (amino acids 42-54) to differentially enhance SR Ca 2؉ release with a nearly 3-fold higher activity of the C terminus. These effects were exclusively based on enhanced SR Ca 2؉ release as S100A1 influenced neither SR Ca 2؉ uptake nor myofilament Ca 2؉ sensitivity/cooperativity in our experimental setting. In conclusion, our study shows for the first time that S100A1 augments contractile performance both of fast-and slow-twitch skeletal muscle fibers based on enhanced SR Ca 2؉ efflux at least mediated by the C terminus of S100A1 protein. Thus, our data suggest that S100A1 may serve as an endogenous enhancer of SR Ca 2؉ release and might therefore be of physiological relevance in the process of excitation-contraction coupling in skeletal muscle.