Modulation of Muscle Contractility by Ancillary Proteins

 

The contractility of striated muscle can be modulated by various mutations in ancillary proteins nebulin, titin and myosin binding protein C (MyBP-C).  These mutations are present in human disease. Several hypotheses have been proposed in order to explain the observations but clear mechanisms of dynamic action of these proteins in the 3-D sarcomere lattice are still lacking. In order to quantitatively assess the effect of these sarcomeric proteins on modulating muscle function, we are developing modules that incorporate these structural proteins in the computational platform, MUSICO.

Three-dimensional structure of sarcomere including regulatory proteins tropomyosin (Tm) and troponin (components TnC, TnI and TnT) and ancillary proteins nebulin and titin.

Simulations of the effect of nebulin on contractility.

 

It is observed that the absence of nebulin results in a broad distribution of thin filament lengths compared to wild type narrow variation of the lengths (Fig. A). Predictions of MUSICO simulations excellently agreed with measured active force in normal (WT) and nebulin deficient (KO) mouse muscles (Fig. B,C). In normal sarcomere geometry with uniform distribution of actin filament lengths (~ 1.2 mm, Fig. A, left) the simulation recapitulated the observed force/pCa curve. In nebulin deficient sarcomere geometry (NDSG) with variable length of actin filaments (Fig. A, right) simulations showed only a modest decrease in isometric force (Fig. B,C). In order to reach the observed decrease of the isometric force in KO muscle, beyond use of the NDSG, it is necessary not only to decrease in      for more than a fourfold, but also significantly decrease muscle sensitivity to calcium; so to match the observations requires increasing the sensitivity of TnI detachment from actin (Fig. C). These results demonstrate that MUSICO simulations could recapitulate force/pCa curves from wild-type and nebulin deficient muscle with only a few adjustments to the model that each represents testable hypotheses.

MUSICO predicted the active force in WT and KO muscles. (A) Actin filament length distributions; (B) Difference in Force-Length and (C) Force-pCa relationships between WT and KO.

The contractility of muscle is modulated by titin.

 

Using the MUSICO platform we quantitatively estimated the effect of titin passive elasticity and change in interfilament spacings on myofilament length dependent activation. The geometrical model of rat atrial trabeculae includes observed variation in actin filament lengths (Fig. A) and nonlinear (passive) elasticity of titin (Fig. B). Large change in length causes change in the filament spacing 3D sarcomere lattice and affects binding kinetices. We compared the active force (per myosin filament) for three different experimental protocols in cardiac muscle: (1) In cardiac sarcomere geometry (CSG) with nonuniform distribution of the length of actin filament filaments (see the distribution in Methods Section), at sarcomere length of 2.3 mm and at high titin tension; (2)  in CSG at sarcomere length of 2.3 mm and low titin tension (mechanical protocol); (3) in CSG at sarcomere length of 2.0 mm.

Predictions active force-pCa relationship by MUSICO platform in cardiac sarcomere geometry with a non-uniform distribution of actin filament lengths, at two sarcomere lengths (2.3 and 2.0 µm) and high and low passive tension. (A) Distribution of actin filament length in rat atrial trabeculae; (B) Force per titin molecule-length relation, black filled symbols with bars are from Helmes at al., 1999, red triangles are from passive sarcomere elasticity at high force, and green symbols at low force; (C) Simulations (small symbols) and Hill curve (solid lines) agree well with observations (symbols with bars) for sarcomere length of 2.3 µm at high and low passive tension, and at sarcomere length of 2 µm.

The predicted titin-dependent tension shows decreases in active isometric force at full activation when fiber shortens below slack length and increases the active force when stretched beyond slack length and increases furthermore at high passive tension (titin). The trend of the force increase is accompanied with increase in sensitivity showing a leftward shift in force-pCa relationship depending on sarcomere length of the degree of titin-dependent passive force (Fig. C). This behavior is caused by partial redistribution of the muscle load between active muscle force and titin-dependent passive force, and also by redistribution of stretch along the thin filament, causing significant changes in TnI-actin bond forces. These changes modulate the release of TnI from actin, i.e. activation of the thin filament. Overall, the data suggest that the Frank-Starling mechanism of the heart may in part be due to an effect of titin-based force on the length dependence of maximal active tension and calcium sensitivity.

Effect of Mutations in cMyBP-C on Sarcomere Mechanical Function.

 

Mutations involving the cMyBP-C gene are known to be the cause of significant cardiac disease in infants, children and adults. In vitro protein-protein binding experiments have demonstrated that mutations in cMyBP-C lead to reduced or failed interaction with sarcomere binding partners, such as the light meromyosin region of myosin (LMM), the S2 region of myosin, F-actin, and titin. The modulated interactions of cMyBP-C with actin or S2, caused by mutations, directly affect cardiac muscle fiber contractility and the cardiac output, and indirectly the muscle genesis during heart development leading to heart hypertrophy. Recent measurements in motility assays provided measurements of kinetics of the interactions between cMyBP-C and actin, serving as a nonproducing crossbridge between myosin and actin filaments and as an inhibitor of myosin binding. In order to assess the effect cMyBP-C mutations on sarcomere contraction we implemented the observed affinities of cMyBP-C to actin in the computational platform MUSICO.

The predicted velocity-force relations of WT vs. cMyBP-C (null) mutant is similar to observations of Korte et al., 2003 for estimated parameters              in (        ) and equilibrium constant       of binding cMyBP-C to actin from motility assay data of Previs et al., 2012 (see motility assays).

Comparison between (predicted) C-zone velocities closely matches observations for WT and cMyBP-C null (mutant) under different conditions and MUSICO predicted velocities in muscle fibers. In both simulations are used best estimates of                in (         ) and equilibrium constant          of binding cMyBP-C to actn from Previs et al., 2012 motility assay data. The predicted velocities in fibers are systematically higher due to different overlap ratios C-zone vs full overlap and three dimensional binding in lattice. The number of attached crossbridges and the number of cMyBP-C per Factin is about 8.4 times higher than in assay because in the lattice three myosin filaments interacting with each actin filaments and higher stability of actomyosin interactions in the 3D lattice.

The predictions from cMyBP-C sarcomeric model showed significant differences between the mutants, and closely follow observations.  This results will allow the substantial re-evaluation of the role of cMyBP-C in the regulation of sarcomere structure and function, the development of a multi-scale myoarchitectural representation of disease phenotype related to the impairment of cMyBP-C binding to various myofilament proteins, and the creation of a novel diagnostic and prognostic methodology for tracking disease progression in patients.

MUSICO predictions of force-pCa WT and cMyBP-C (null) muscle fibers. Symbols are the MUSICO predictions and lines are Hill curves. The predicted rations are similar to observations of Witt et at., 2001.