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.)
SUMMARY1. Ca2+ currents in frog skeletal muscle fibres were studied with a voltage-clamp technique. Under membrane depolarization maintained for several seconds, Ca2+ current was found to decline with time constants of 0-2-2 see when LCa2+]o = 10 mM.2. Ca2+ currents are diminished by nifedipine, D-600, tetracaine and Ni2+.3. When peak current is diminished by making the membrane potential positive, by block with drugs or by substituting the relatively less permeant Mn2+ for Ca2+, then the rate ofdecline is diminished also. When peak current is increased by recording at relatively negative membrane potentials or by substituting for Ca2+ the more permeant ions Ba2+ or Sr2+, then the rate of decline is increased in proportion. Evidently, the size of the current determines the rate of decline.4. Decline of current is greatly slowed in isotonic Ca2+ saline or when the [Ca2+]o is buffered by the organic anion malate. These findings indicate that the decline of current arises from Ca2+ depletion in an extracellular compartment, most probably the transverse tubules. On this basis, an analysis of Ca2+ current decline and recovery leads to the following conclusions.5. Ca2+ current flows almost entirely across the membranes of the transverse tubules.6. After allowing for the tortuosity of the tubular network, the apparent diffusion coefficient for Ca2+ in the transverse tubules is about 2-6 x 10-6 cm2/sec, three times less than the diffusion coefficient for K+ in the transverse tubules and about three times less than the diffusion coefficient for Ca2+ in free solution.7. The transverse tubule lumen does not appear to have a large Ca2+-buffering capacity in the millimolar range. At [Ca2+]j = 10 mM, the tubule lumen binds less than 0-6 dissociable Ca2+ ions for every free ion.
Progressive force loss in Duchenne muscular dystrophy is characterized by degeneration/regeneration cycles and fibrosis. Disease progression may involve structural remodeling of muscle tissue. An effect on molecular motorprotein function may also be possible. We used second harmonic generation imaging to reveal vastly altered subcellular sarcomere microarchitecture in intact single dystrophic mdx muscle cells (approximately 1 year old). Myofibril tilting, twisting, and local axis deviations explain at least up to 20% of force drop during unsynchronized contractile activation as judged from cosine angle sums of myofibril orientations within mdx fibers. In contrast, in vitro motility assays showed unaltered sliding velocities of single mdx fiber myosin extracts. Closer quantification of the microarchitecture revealed that dystrophic fibers had significantly more Y-shaped sarcomere irregularities ("verniers") than wild-type fibers (approximately 130/1000 microm(3) vs. approximately 36/1000 microm(3)). In transgenic mini-dystrophin-expressing fibers, ultrastructure was restored (approximately 38/1000 microm(3) counts). We suggest that in aged dystrophic toe muscle, progressive force loss is reflected by a vastly deranged micromorphology that prevents a coordinated and aligned contraction. Second harmonic generation imaging may soon be available in routine clinical diagnostics, and in this work we provide valuable imaging tools to track and quantify ultrastructural worsening in Duchenne muscular dystrophy, and to judge the beneficial effects of possible drug or gene therapies.
SUMMARY1. A study was carried out to investigate the effects of ionic strength and monovalent cations on isometric, Ca2+-activated force and rigor responses in mechanically skinned muscle fibres. Three types of skeletal muscle fibres were used: rat fast-and slow-twitch fibres and toad twitch fibres.2. The contractile apparatus of rat slow-twitch fibres was affected differently from that of rat fast-twitch and amphibian twitch fibres when changing the ionic strength (expressed either in terms of ionic equivalents as I or formally as F/2) and [K+]. Thus, the apparent sensitivity to Ca2+ decreased substantially more in slow-twitch fibres (by a factor of 20) than in the other fibre types (by a factor of 12) when I and [K+] were increased from 94 to 354 mm and from 56 to 316 mm respectively. Maximum Ca2+-activated force, however, declined only by a factor of 2-2 in slow-twitch fibres compared with 4-2 in the other fibre types, when I was increased from 154 to 354 mm.3. In slow-twitch fibres the force oscillations of myofibrillar origin were found to increase substantially in amplitude, duration and frequency at low values of I and almost disappeared at high ionic strength. At low values of I, it was also discovered that ca. 50 % of the fast-twitch fibres responded with myofibrillar force oscillations when submaximally activated. The characteristics of these oscillations were different from those of slow-twitch fibres.4. Rigor force levels were found to decline markedly with increasing I and [K+] in all fibre types. Unexpectedly, once rigor force was established in a certain ionic environment, the level of force was stable regardless of further changes in ionic strength and monovalent cation concentration. These results indicate that the rigor cross-bridges can be formed in different stable positions and that the probability of attachment in certain positions (rather than the total number of cross-bridges that can be formed) is influenced by the ionic conditions. 5. Further experimental evidence provided in this study shows that the increase in [K+] is mainly responsible for the decrease of the Ca2+-sensitivity of the contractile apparatus and that ionic strength (expressed as I rather than F/2) influences markedly (i) the maximal Ca2+-activated force, (ii) the maximum steepness of the pCa-force relations and (iii) the oscillatory processes of myofibrillar origin.
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.
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