Molecular Mechanisms of Deregulation of Muscle Contractility Caused by the R168H Mutation in TPM3 and Its Attenuation by Therapeutic Agents

Abstract

The substitution for Arg168His (R168H) in γ-tropomyosin (TPM3 gene, Tpm3.12 isoform) is associated with congenital muscle fiber type disproportion (CFTD) and muscle weakness. It is still unclear what molecular mechanisms underlie the muscle dysfunction seen in CFTD. The aim of this work was to study the effect of the R168H mutation in Tpm3.12 on the critical conformational changes that myosin, actin, troponin, and tropomyosin undergo during the ATPase cycle. We used polarized fluorescence microscopy and ghost muscle fibers containing regulated thin filaments and myosin heads (myosin subfragment-1) modified with the 1,5-IAEDANS fluorescent probe. Analysis of the data obtained revealed that a sequential interdependent conformational-functional rearrangement of tropomyosin, actin and myosin heads takes place when modeling the ATPase cycle in the presence of wild-type tropomyosin. A multistep shift of the tropomyosin strands from the outer to the inner domain of actin occurs during the transition from weak to strong binding of myosin to actin. Each tropomyosin position determines the corresponding balance between switched-on and switched-off actin monomers and between the strongly and weakly bound myosin heads. At low Ca2+, the R168H mutation was shown to switch some extra actin monomers on and increase the persistence length of tropomyosin, demonstrating the freezing of the R168HTpm strands close to the open position and disruption of the regulatory function of troponin. Instead of reducing the formation of strong bonds between myosin heads and F-actin, troponin activated it. However, at high Ca2+, troponin decreased the amount of strongly bound myosin heads instead of promoting their formation. Abnormally high sensitivity of thin filaments to Ca2+, inhibition of muscle fiber relaxation due to the appearance of the myosin heads strongly associated with F-actin, and distinct activation of the contractile system at submaximal concentrations of Ca2+ can lead to muscle inefficiency and weakness. Modulators of troponin (tirasemtiv and epigallocatechin-3-gallate) and myosin (omecamtiv mecarbil and 2,3-butanedione monoxime) have been shown to more or less attenuate the negative effects of the tropomyosin R168H mutant. Tirasemtiv and epigallocatechin-3-gallate may be used to prevent muscle dysfunction.

Tropomyosin 3 (TPM3) function in skeletal muscle and in myopathy

“…However, for several other mutations, there exists no such linear relationship. Substitutions R168H, E151A, and K169E have been associated with reduced or increased Ca 2+ sensitivity in different studies [ 46 , 131 , 137 , 140 , 142 , 144 – 146 ]. This may indicate the complexity of mature fibers in which the mechanical properties of myofilaments not only depend on tropomyosin but also on other factors which may have adapted to disease.…”

“…Pharmacological strategies targeting thin and thick filaments aim at reversing contractile dysfunction at the sarcomere level. For instance, the use of troponin activators (e.g., EMD 57033, tirasemtiv), troponin inhibitors (e.g., epigallocatechin-3-gallate, W7), myosin activator (e.g., Omecamtiv Mecarbil) and myosin inhibitor (e.g., BDM) partially reverse contractile defects in patient muscle fibers and reconstructed thin filaments in vitro [ 134 , 140 , 141 , 144 ]. In these studies, diverse effects on contractile properties are observed depending on the strategy used and the mutation.…”