Exchange of β- for α-Tropomyosin in Hearts of Transgenic Mice Induces Changes in Thin Filament Response to Ca2+, Strong Cross-bridge Binding, and Protein Phosphorylation

Abstract

Despite its potential as a key determinant of the functional state of striated muscle, the impact of tropomyosin (Tm) isoform switching on mammalian myofilament activation and regulation in the intact lattice remains unclear. Using a transgenic approach to specifically exchange ␤-Tm for the native ␣-Tm in mouse hearts, we have been able to uncover novel functions of Tm isoform switching in the heart. The myofilaments containing ␤-Tm demonstrated an increase in the activation of the thin filament by strongly bound cross-bridges, an increase in Ca 2؉ sensitivity of steady state force, and a decrease in the rightward shift of the Ca 2؉ -force relation induced by cAMP-dependent phosphorylation. Our results are the first to demonstrate the specific effects of Tm isoform switching on mammalian thin filament activation in the intact lattice and suggest an important role for Tm in modulation of myofilament activity by phosphorylation of troponin.The ability of myosin heads to react with actin in heart muscle occurs with a transition of the thin filament from an "off" to an "on" state that depends on complex alterations involving the tropomyosin (Tm) 1 molecule (for reviews see Refs. 1 and 2). These alterations include possible steric effects associated with changes in the position of Tm on the thin filament, as well as allosteric and cooperative effects associated with Tminduced changes in actin structure and reactivity with myosin (3-5 -TnC itself cannot activate the thin filament, but acts as a co-factor shifting the equilibrium between off and on states of Tm such that strongly bound cross-bridges more easily activate the thin filament. Although recent considerations indicate that activation may involve both processes (2), the relative role of the steric and allosteric/cooperative mechanisms in turning on the activity of striated muscle remains unclear.Our perception of the role of Tm in the regulation of striated muscle, as well as it's structure/function relations, has come from a variety of approaches. These include x-ray diffraction of muscle preparations (7) and crystals (8, 9), reconstructions from electron micrographs (9, 10), and reconstitution studies of soluble systems with Tm (11-13), Tm peptides (14), and mutants of Tm (15). In some cases, inferences regarding structure/ function relations have been made from comparisons of muscle fibers containing isoforms of Tm (16). However, interpretation of these studies is difficult in that there are multiple changes in myofilament proteins that occur along with the natural variations in Tm. A clearer understanding of the structure/function relations of Tm has been hampered by an apparent lack of methods for reversibly extracting Tm from the myofilament lattice in a force-generating system, as has proved so successful in the case of Tn components such as TnC and TnI (17). Thus, issues such as the role of Tm domains, covalent modifications, and the functional significance of isoform switching of Tm in the intact force-generating lattice of vertebrate-striated muscle ...

Myofibrillar remodelling in cardiac hypertrophy, heart failure and cardiomyopathies

“…A decrease in the α-TM to β-TM ratio (from the normal 49:1 ratio to 1:1), was associated with diastolic dysfunction (197), increased Ca 2+ sensitivity in skinned fibre preparations (198) and decreased maximal rates of contraction and relaxation in isolated cardiomyocytes (199). A markedly increased expression of β-TM in mouse hearts (α-TM to β-TM ratio of approximately 1:4) leads to the death of animals soon after birth, along with severe cardiac abnormalities in both contraction and relaxation (197).…”

Patterns of tropomyosin and troponin-T isoform expression in jaw-closing muscles of mammals and reptiles that express masticatory myosin

“…Differences in Ca 2+ -sensitivity or cooperativity of force generation of striated muscle are associated with different isoforms of Tm and TnT (e.g. Schachat et al, 1987;Greaser et al, 1988;Palmiter et al, 1996;Gallon et al, 2006;McCall et al, 2006;Jagatheesan et al, 2009). It is possible, therefore, that significant differences in the Ca 2+ regulation of force generation between jaw-closing and limb muscles within individual species and in jaw-closing muscles between species are associated with the differences in the Tm and TnT isoform expression patterns observed in this study.…”

“…Different isoforms of TnT and Tm are associated with variations in the Ca 2+ -sensitivity or cooperativity of force generation in muscle, as determined by shifts in the force- [Ca 2+ ] relationship in fibers in which the surface membrane is removed or rendered hyperpermeable (e.g. Schachat et al, 1987;Greaser et al, 1988;Palmiter et al, 1996;Ogut et al, 1999;McCall et al, 2006;Biesiadecki et al, 2007;Jagatheesan et al, 2009). Furthermore, Tm isoforms can differentially modulate contractile properties through the TnT-binding domain, independent of differences in Ca 2+ -sensitivity (Jagatheesan et al, 2004).…”

An internal domain of β-tropomyosin increases myofilament Ca2+sensitivity

“…Data were normalized to the percent maximum tension at 2.3 m of sarcomere length with the maximum tension of the ␣-TM NTG control taken as 100%. The various pCa-percent maximal force graphs were derived from the following distinct TG models: ␣-TM is the NTG control; ␤-TM is the overexpression of the complete ␤-TM isoform (18,21,33); ␣-/␤-TM1 is the overexpression of the chimeric TM protein encoding amino acids 1-257 of ␣-TM ligated to amino acids 258 -284 of ␤-TM (12); ␣-/␤-TM2 is the overexpression of the chimeric TM protein encoding amino acids 1-174 of ␣-TM, amino acids 175-190 of ␤-TM, amino acids 191-257 of ␣-TM, and amino acids 258 -284 of ␤-TM (10); and ␣-/␤-TM3 is the TG model described in this work. Fig.…”