Chien-Kao Wang

Thin filament near-neighbour regulatory unit interactions affect rabbit skeletal muscle steady-state force-Ca2+ relations

The role of cooperative interactions between individual structural regulatory units (SUs) of thin filaments (7 actin monomers : 1 tropomyosin : 1 troponin complex) on steady‐state Ca2+‐activated force was studied. Native troponin C (TnC) was extracted from single, de‐membranated rabbit psoas fibres and replaced by mixtures of purified rabbit skeletal TnC (sTnC) and recombinant rabbit sTnC (D27A, D63A), which contains mutations that disrupt Ca2+ coordination at N‐terminal sites I and II (xxsTnC). Control experiments in fibres indicated that, in the absence of Ca2+, both sTnC and xxsTnC bind with similar apparent affinity to sTnC‐extracted thin filaments. Endogenous sTnC‐extracted fibres reconstituted with 100 % xxsTnC did not develop Ca2+‐activated force. In fibres reconstituted with mixtures of sTnC and xxsTnC, maximal Ca2+‐activated force increased in a greater than linear manner with the fraction of sTnC. This suggests that Ca2+ binding to functional Tn can spread activation beyond the seven actins of an SU into neighbouring units, and the data suggest that this functional unit (FU) size is up to 10–12 actins. As the number of FUs was decreased, Ca2+ sensitivity of force (pCa50) decreased proportionally. The slope of the force‐pCa relation (the Hill coefficient, nH) also decreased when the reconstitution mixture contained < 50 % sTnC. With 15 % sTnC in the reconstitution mixture, nH was reduced to 1.7 ± 0.2, compared with 3.8 ± 0.1 in fibres reconstituted with 100 % sTnC, indicating that most of the cooperative thin filament activation was eliminated. The results suggest that cooperative activation of skeletal muscle fibres occurs primarily through spread of activation to near‐neighbour FUs along the thin filament (via head‐to‐tail tropomyosin interactions).

Ca2+ Regulation of Rabbit Skeletal Muscle Thin Filament Sliding: Role of Cross-Bridge Number

We investigated how strong cross-bridge number affects sliding speed of regulated Ca(2+)-activated, thin filaments. First, using in vitro motility assays, sliding speed decreased nonlinearly with reduced density of heavy meromyosin (HMM) for regulated (and unregulated) F-actin at maximal Ca(2+). Second, we varied the number of Ca(2+)-activatable troponin complexes at maximal Ca(2+) using mixtures of recombinant rabbit skeletal troponin (WT sTn) and sTn containing sTnC(D27A,D63A), a mutant deficient in Ca(2+) binding at both N-terminal, low affinity Ca(2+)-binding sites (xxsTnC-sTn). Sliding speed decreased nonlinearly as the proportion of WT sTn decreased. Speed of regulated thin filaments varied with pCa when filaments contained WT sTn but filaments containing only xxsTnC-sTn did not move. pCa(50) decreased by 0.12-0.18 when either heavy meromyosin density was reduced to approximately 60% or the fraction of Ca(2+)-activatable regulatory units was reduced to approximately 33%. Third, we exchanged mixtures of sTnC and xxsTnC into single, permeabilized fibers from rabbit psoas. As the proportion of xxsTnC increased, unloaded shortening velocity decreased nonlinearly at maximal Ca(2+). These data are consistent with unloaded filament sliding speed being limited by the number of cycling cross-bridges so that maximal speed is attained with a critical, low level of actomyosin interactions.

Characterization of troponin-C interactions in skinned barnacle muscle: comparison with troponin-C from rabbit striated muscle

Previously it was shown that when troponin-C (TnC) is extracted from barnacle myofibrillar bundles they lose their Ca2+ sensitivity, which can be restored by adding back barnacle TnC (either isoform, BTnC1 or BTnC2). Thus barnacle muscle shows thin filament regulation, as does rabbit psoas skeletal muscle. In this paper we comp are the interactions of barnacle and rabbit fast muscle TnC in their respective muscles. We demonstrate that muscle fibres from the giant barnacle, Balanus nubilus, contain about 186 μm kg−1 muscle tissue of BTnC1 plus BTnC2 compared to about 91 μm kg−1 of TnC in rabbit psoas muscle fibres. Extraction of BTnC is achieved using similar low ionic strength, low divalent ion Ca2+-low Mg2+ conditions which are required for TnC extraction in rabbit psoas skinned muscle fibres; extraction was prevented by 1 mm Mg2+. Full reconstitution of Ca2+-sensitivity was achieved by adding back BTnC (1 + 2, or 2). Reconstitution of barnacle muscle with rabbit fast skeletal TnC (RTnC) was more complex, with partial recovery of Ca2+-sensitivity with reconstitution in the presence of 3 mm Mg2+ and more fully with reconstitution in the presence of activating Ca2+ (pCa 4.0). This suggests that the barnacle TnC-TnI (troponin I) recognition sites may be more complex than in rabbit because the barnacle sites appear to have at least two different conformations or types, in which one recognizes RTnC in the presence of Mg2+ and the other only in the presence of Ca2+ and Mg2+. This is consistent with the presence of several TnI isoforms in barnacle striated myofibrils. RTnC has two C-terminal Ca2+-Mg2+ binding sites that are thought to be involved in the Mg2+-sensitive binding of RTnC in rabbit muscle, yet it has been suggested that this site in barnacle muscle does not bind Mg2+, even though Mg2+ stabilizes BTnC binding in barnacle muscle. Consistent with this stabilizing action of Mg2+, using fluorescent probes IAANS or IAE on isolated BTnC2 we demonstrate that BTnC2 binds both Ca2+ and Mg2+, but the data do not suggest direct competition. Consistent with the C-terminal sites on BTnC being Ca2+-specific, BTnC1+2 could only reconstitute low levels of force (about 1/3) in TnC-extracted rabbit skinned muscle fibers in the presence of pCa 4.0 (not just Mg2+) and only at low ionic strengths (0.09 m). Ca2+-activation of contraction was further examined using fluorescently labelled BTnC2 (labelled with IANBD) incorporated into skinned barnacle myofibrillar bundles. Maximal Ca2+ binding produced structural changes in BTnC which resulted in a 45% decrease in the fluorescence compared to the value at pCa 9.2. The magnitude of the fluoresence decrease paralleled the increase in force with increas ing Ca2+. The Hill fits to the data gave pCa1/2 and n of 5.61 ± 0.02 and 2.06 ± 0.12 for force, and 5.52 ± 0.02 and 1.88 ± 0.10 for fluoresence. Removing MgATP to induce rigor in the fibre decreased BTnC2-NBD fluorescence only about 11%, but the addition of Ca2+ in rigor further decreased the fluorescence to a slightly larger extent than under maximal Ca2+ activating conditions. These fluorescence changes are qualitatively similar to the fluorescence enhancement seen with Ca2+-activation and rigor with RTnCDanz exchanged into rabbit psoas skinned muscle fibres. The data support a similar model for Ca2+-activation of force in barnacle muscle and in rabbit psoas skeletal muscle fibres

Cooperative Activation of Skeletal and Cardiac Muscle

Both skeletal and cardiac muscles show a steep force-pCa relationship indicative of cooperative activation, but there are differences in some of the underlying mechanisms of this cooperativity. As we have discussed previously (Gordon et al, 2000), these give rise to significant differences in the properties of skeletal and cardiac muscle that are important for their various physiological roles and methods of control. Cardiac contractions occur spontaneously and rhythmically, driven by the cardiac pacemaker cells in the SA node, with spread of electrical activity from cell to cell. This activates cardiac cells in sequence to eject blood allowing the heart to function as a periodic pump. Since each cell contracts on each beat, variations in cardiac output occur with variations in heart rate and the strength of contraction on each beat. Through intrinsic and extrinsic regulation via the autonomic nervous system, the rate and strength of each contraction can be regulated to meet the circulatory needs. In contrast, skeletal muscle contraction is controlled through the central nervous system as motor units, defined as a motoneuron and the muscle fibers it innervates. Although force varies with frequency of stimulation of each motor unit, the major means of regulation is by recruitment of motor units, a mechanism unavailable to the heart cells.

Effect of phosphorylation of cardiac troponin I on the fluorescence properties of its single tryptophan as determined by picosecond spectroscopy

Cardiac troponin I (CTnI) can be phosphorylated by a c-AMP dependent protein kinase. We have investigated the effect of the phosphorylation on the emission decay properties of its single tryptophan by using a cavity dumped and synchronously pumped dye laser system. At 20°C, τ1~0.60 ns, τ2~2.22 ns, and τ3~4.72 ns. The corresponding fluorescence contributions were 7%, 47%, and 46%, respectively. Upon phosphorylation four lifetimes were observed: τ1~0.11 ns, τ2~0.81 ns, τ3~1.95 ns, and τ4~6.63 ns, and fractional contributions of the four components were 2%, 16%, 52%, and 30%, respectively. This finding indicates that the environment of the tryptophan is modified by phosphorylation. In the absence of divalent metal ions, the observed three decay times of the CTnI complexed with cardiac troponin C (CTnC) remained unchanged, and addition of Ca2+ or Mg2+ resulted in only small changes in the lifetimes. When phosphorylated CTnI was complexed with CTnC, a large increase of the longest-lived component was observed: τ4 > 11 ns with its contribution shifted to 47%. Two rotational correlation times were observed for CTnI: φ1~0.9 ns and φ2~ 23.5 ns. These valves increased to t0~l.2 ns and ~30.1 ns, respectively, for the complex CtnI CTnC. Upon phosphorylation the two correlation times were significantly reduced regardless of whether CTnI was uncomplexed or complexed with CTnC. These results suggest that phosphorylation of CTnI resulted in a significantly more compact structure and enhanced motion of the tryptophan side chain. These structural changes may play a role in the transmission of Ca2+ signal in cardiac muscle. Thus, the effect of phosphorylation of CTnI becomes more pronounced when the protein is complexed with CTnC. These results suggest that there was likely a fluorophore heterogeneity which may arise from differences in conformation, environment, and/or different deactivation pathways for the excited state of the fluorophore.

Time-resolved fluorescence studies on the single tryptophan in bovine brain S-100a protein

The fluorescence emission of the single tryptophan in bovine brain S-lOOa protein has been studied by using time-resolved laser fluorescence spectroscopy. The tryptophan fluorescence emission was isolated by a Schott 0-54 cut-off filter at right angles to the excitation direction by a Hamamatsu R955P photomultiplier. With excitation at 295 nm, the fluorescence decay of S-lOOa protein in 25 mM Tris buffer was best represented by a sum of three exponential terms regardless of solvent conditions. At 20°C and pH 7.2, the three components of lifetime for apo-S-lOOa protein were (tau)1~0.43 ns, (tau)2~1.24 ns, and (tau)3~4.05 ns. The corresponding fluorescence contributions of each component were 31%, 31% and 38%, respectively. When the protein was saturated with Mg2, the lifetimes increased slightly and the contribution of the shortest fluorescence component ((tau)1) to the total emission intensity increased slightly at the expense of the other two components. Binding of Ca2+ to S-lOOa protein resulted in a significant decrease of (tau)1, and a substantial increase of (tau)2 and (tau)3, and an increase of the average of the three lifetimes. Under this condition the fractional fluorescence contributions associated with (tau)2 and (tau)3 increased very significantly at the expense of the shortest component. The fluorescence decay behavior of each of he S-lOOa samples was relatively insensitive to the variation of temperature (4~20°C). At pH 8.2 and pH 8.4, the decay behavior of the protein in response to binding of Ca2+ and Mg2+, and changes of temperature was very similar to those observed at pH 7.2. The triple exponential decay kinetics of the single tryptophan in S-lOOa protein could be rationalized by the existence of multiple local conformers.