Nebulin and titin modulate cross-bridge cycling and length-dependent calcium sensitivity

Figures

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  Figure 1.. The 3-D structural organization of sarcomeres includes thick (or myosin) filaments with interdigitated thin filaments composed of actin and regulatory proteins and the ancillary proteins nebulin and titin. Thin filaments are cross-linked within the Z-disc by protein networks at both ends of a sarcomere, whereas the myosin filaments are cross-linked in the M-line situated in the middle of the filaments and in the central region of the sarcomere. Nebulin (pink line) spans most of the length of thin filament, with its C terminus in the Z-disc and its N terminus near the thin filament pointed end. Enlarged portion of actin filament shows capping protein tropomodulin (Tmod), tropomyosins (Tms), and components of the Tn complex: TnC with two or one calcium binding sites as expressed in skeletal and cardiac muscles, respectively; TnI interacting with actin to anchor associated Tm to actin filaments in the absence of bound calcium; and TnT supporting head-to-tail interactions between two neighboring Tm molecules to form a CFC. Each Tm molecule covers seven actin monomers and is associated with one Tn complex, also repeating every seventh actin monomer. Two CFCs span the length of thin…

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  Figure 2.. 3-D view of myosin (thick) filaments in vertebrate striated muscle. (A) Myosin filaments extend from the central M-band toward Z-discs with cross-bridges arranged in a three-stranded helical arrays with helical pitches of 3 × 42.9 nm, i.e., three myosin molecules (crowns) are located at 14.3-nm intervals. Inspired by 3-D lattice figure from Williams et al. (2013). (B) The crowns, consisting of three myosin molecules, are shifted 40° azimuthally axially for 14.3 nm and repeating every fourth crown displaced axially for 42.9 nm. Further away, myosin molecules are shown in lighter colors. Inspired by projection of myosin filament in the lattice cross section from figure in Squire (1997).

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  Figure 3.. The relative distance between a myosin head and the adjacent binding site on actin in the 3-D sarcomere lattice. (A) Myosin molecules form a triple helix along the myosin filament where the myosin heads are arranged in layers and at each layer form a “crown” with three myosin molecules. The crowns Lc = 1, 2, and 3 are axially separated by 14.3 nm and rotated by 40°, forming different angular arrangements with actin filaments, but only those that might interact with the actin filament are shown. On the other side, the actin monomers are helically arranged in a double-strand helical structure, and favorable myosin-binding sites on actin filament associated with nearby myosin heads are shown in dark gray. In the axial direction, each myosin pair of heads, denoted as a cross-bridge and multiple binding sites on surrounding actin filaments, forms a large number of arrangements defined by the change in relative axial distances, x, between the unstrained position of the cross-bridge and the nearest actin-binding site. (B) The hexagonal sarcomere lattice with 2:1 actin-to-myosin-filament ratio shows in the azimuthal plane that up to three myosins can attach to each actin filament. The…

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  Figure 4.. Nebulin specifies thin filament length distributions in skeletal muscle. (A and B) The distance between silver grains and the edge nearest the Z-disc, denoted as Z-line, from mouse tibialis cranialis muscle, observed by Witt et al. (2006), shows uniform distribution of grains in WT, up to a distance of 1.2 µm (A), in contrast, to the grain distribution in Neb-KO muscle fibers, which gradually decreases from ∼0.4 to ∼1.2 µm (B). The red lines are best fits of the histograms in A and B by a function Φc(Lz) relating observed frequency of silver grains at distance from Z-line, Lz. (C and D) After normalization of Φc(Lz) with maximum frequency a and setting thin filament length as La = Lz, the cumulative probability function of filament lengths Φ^ (red lines) and thin filament length distributions dΦ^/dLa (green lines) are formulated and displayed for WT (C) and Neb-KO (D) muscle fibers. Spatially distributed actin filaments of variable lengths across a sarcomere, necessary for MUSICO simulations, can be obtained from sequentially drawn random numbers, Rnd#, in a range from 0 to 1 (D).

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  Figure 5.. Distribution of actin (thin) filament lengths in rat atrial trabeculae and description of titin’s nonlinear elasticity. (A) The best fit of the thin filament length distribution (green line) observed in rat atrial trabeculae (gray bars; Robinson and Winegrad, 1977). (B) The cumulative probability density function Φ^ (red line) is derived from the best fit (green line) and used for generation of spatially distributed lengths of thin filaments in sarcomere lattice by sequentially drawing random numbers, 0≤Rnd#≤1. Derived sarcomere geometry mimics the distributed thin filament lengths observed in the atrial trabeculae, and it is used in the MUSICO simulations. (C) The nonlinear elasticity of a titin molecule as a function of SL observed in cardiac muscle by Helmes et al. (1999) (black filled symbols with bars). The force per titin molecule estimated from HPT (Cazorla et al., 2001) at two SLs (red filled triangle) matches the force–length data reported by Helmes et al. (1999). In contrast, the force at LPT (mechanical) underestimates the observed values at all three SLs (cyan-filled blue triangles). Sarcomere lattice compression with 4% dextran did not affect titin forces, showing…

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  Figure 6.. The effect of nebulin on force–length and force–pCa relations. (A) The MUSICO-predicted force–SL relation (pink triangles and line) agrees well with observations in WT muscle (green circles with bars). Including in calculations only the variation of thin filament lengths in Neb-KO (gray squares and line) shows an ∼18% decrease in force at full overlap (WT at SL = 2.5 µm) and shifts to the left by ∼0.4 µm. To achieve the observed large drop in active tension (red triangles with bars), it was necessary to decrease the affinity of myosin to regulated thin filament (i.e., kbind) by ∼2.4-fold, to bring predictions (cyan-filled blue triangles and line) close to observations in Neb-KO. (B) Predicted force–pCa relation at 2.5 µm for WT (pink triangles and line) agrees well with observations (green filled circles with bars; Witt et al., 2006) and, at fully activated muscle with the nebulin-deficient sarcomere geometry and the same change in binding affinity as used in A, was sufficient to simulate magnitude of force at high calcium concentrations (blue open triangle and blue dotted line), but the predictions showed lower calcium sensitivity for ΔpCa50 = 0.0898 (horizontal blue arrow). An…

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  Figure 7.. Effect of titin (passive) force on activation. (A–C) Observed force–pCa relations (symbols with bars) are compared with MUSICO predictions (symbols only) at SL 2.0 and 2.3 µm and at high and low tension. HPTs are achieved by stretching myocytes from SL 2.0 to 2.3 µm and then activating. Low tension is achieved by stretching myocytes to SL 2.5 µm, holding, releasing to SL of 2.3 µm, and then activating (mechanical protocol; A); low tension was also achieved by trypsin treatment of the cells at SL 2.3 µm (B); the effect of osmotic compression (2.5% dextran; C). For reference, thin gray lines in B and C show force–pCa relations at 2.3 µm SL at HPT and at 2.0 µm SL from A.

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  Figure 8.. Effect of SL on isometric force in fully activated cardiac muscle. Changes of multiple factors are shown at SLs 2.3, 2.0, and 1.9 µm. Thin-thick overlap increases with the decrease of SL. The overlap is displayed as a fraction of thick-thin overlap at full overlap, where average thin filament length (dashed lines) display the magnitude of the average overlap. The binding rate, kbind, decreases with the decrease of SL. The thick-thin filament overlap defines the region where crossbridges can bind to thin filaments and reflects the number of bound crossbridges per half myosin filament and, therefore, developed isometric force and tension. The titin-based passive tension is high at SL = 2.3 µm, low at SL = 2.0 µm, and ∼0 at SL = 1.9 µm. The interfilament spacing increases with the decrease in SL. The values shown in bar graphs are as follows: at SL = 2.3 µm as green bars, at 2.0 µm as orange bars, and at 1.9 µm as red bars. kbind and active tensions are is shown as percentages of the values at SL = 2.3 µm. The thin-thin filament overlap is small, and only a small number of thin filaments interfere; thus its contribution to decrease in active force or tension is negligible.