Enhanced Active Cross-Bridges during Diastole: Molecular Pathogenesis of Tropomyosin's HCM Mutations

Abstract

Three HCM-causing tropomyosin (Tm) mutants (V95A, D175N, and E180G) were examined using the thin-filament extraction and reconstitution technique. The effects of Ca(2+), ATP, phosphate, and ADP concentrations on cross-bridge kinetics in myocardium reconstituted with each of these mutants were studied at 25°C, and compared to wild-type (WT) Tm at physiological ionic strength (200 mM). All three mutants showed significantly higher (2-3.5 fold) low Ca(2+) tension (T(LC)) and stiffness than WT at pCa 8.0. High Ca(2+) tension (T(HC)) was significantly higher for E180G than that for WT, whereas T(HC) of V95A and D175N was similar to WT; high Ca(2+) stiffness (Y(HC)) had the same trend. The Ca(2+) sensitivity of isometric force was significantly greater for V95A and E180G than for WT, whereas that of D175N remained the same as for WT; for all mutants, cooperativity was lower than for WT. Nine kinetic constants and the cross-bridge distribution were deduced using sinusoidal analysis. The number of force-generating cross bridges was similar among the D175N, E180G, and WT Tm forms, but it was significantly larger in the case of V95A than WT. We conclude that the increased number of actively cycling cross bridges at pCa 8 is the major cause of Tm mutation-related HCM pathogenesis, which may result in diastolic dysfunction. Decreased contractility (T(act)) in V95A and D175N may further contribute to the severity of myocyte hypertrophy and related prognosis of the disease.

Mechanistic Heterogeneity in Contractile Properties of α-Tropomyosin (TPM1) Mutants Associated with Inherited Cardiomyopathies

“…TnT mutants that showed no change in sliding velocity when tested with human ␤-cardiac myosin showed significant changes when tested with non-human myosins (33). The D175N HCM Tm mutant produced no change in Ca 2ϩ sensitivity when assayed in rat or bovine contexts (51,52), but it increased Ca 2ϩ sensitivity when assayed using rabbit fast skeletal heavy meromyosin (53). When the R453C HCM mutant was produced in mouse ␣-cardiac MHC (54) and human ␤-cardiac MHC (55), there was a similar increase in single-molecule force production.…”

Patterns of Disease Progression in Hypertrophic Cardiomyopathy

“…26 Additional disease mechanisms involve impairment of mechanisms that switch off contraction at low cytosolic [Ca 2+ ], leading to incomplete relaxation and diastolic dysfunction while increasing energetic compromise. [26][27][28] Chronic dysregulation of cardiomyocyte Ca 2+ homeostasis may cause multiple downstream effects involving secondary activation of Ca 2+ -regulated signaling pathways, cardiac remodeling and, possibly, apoptosis. Furthermore, sarcomeres and their Z-disk components are now recognized centers of mechano-sensation, mechanotransmission, and mechano-transduction.…”

Multi-scale computational models of familial hypertrophic cardiomyopathy: genotype to phenotype

“…Multi-scale FHC models S. G. figure 3a, where the model is used to reproduce a leftward shift and loss of cooperativity in the force -Ca 2þ relation seen in the FHC-linked tropomyosin mutation E180G [17]. Both changes were achieved simultaneously by lowering a single parameter, the free energy change associated with nearest-neighbour interactions between tropomyosins.…”

“…Traditionally, EC coupling has been considered complete at the point Ca 2þ binds to troponin C (TnC), but two observations suggest that this view neglects potentially important mechanisms in the behaviour of cardiac muscle. The first is that the Ca 2þ affinity of TnC is increased nearly 10-fold by myosin (a) Qualitative changes in the steady-state force-pCa relation owing to the Tm mutation E180G [17], including an increase in Ca 2þ sensitivity and decrease in cooperativity (steepness of the curve) were recapitulated in a myofilament model [23] by reducing the amount of nearest-neighbour Tm coupling (control and E180G Tm are solid and dashed lines, respectively). This parameter change is indicative of either increased Tm flexibility or destabilization of end-to-end binding.…”

“…This means that, during a twitch, the capacity of TnC as a buffer of cytosolic Ca 2þ cannot be considered constant. A second general observation is that, in numerous cases, modification of myofilament proteins alters the overall Ca 2þ sensitivity (and presumably Ca 2þ buffering) of the sarcomeres, whether through post-translational modification [37] or cardiomyopathy-linked mutations [17]. The abundance of TnC in the cytosol means that factors modifying myofilament Ca 2þ sensitivity have the potential to influence EC coupling in the whole cell, one form of a phenomenon known as mechanoelectric feedback [38].…”