Green tea catechin EGCG is able to partially restore the regulation of muscle contraction by the troponin-tropomyosin complex, impaired by the Glu150Ala substitution in γ-tropomyosin

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Abstract

A number of point mutations has been identified in the genes of contractile and regulatory proteins of skeletal muscle that can lead to dysfunction of muscle tissue. The molecular mechanisms of muscle contraction in the presence of mutant muscle proteins in the sarcomere remain poorly understood. In the current study, we examined the impact of the glutamate-to-alanine substitution at position 150 (Glu150Ala) of γ-tropomyosin associated with cap disease and fiber-type disproportion in humans on the molecular mechanisms of troponin-tropomyosin-related regulation of muscle contraction in a single muscle fiber. It is believed that tropomyosin residue Glu150 is not directly involved in the interaction of tropomyosin with actin and myosin interactions; however, according to structural models of thin filaments under low Ca2+ conditions, this residue is located close to site of binding with the C-terminal domain of troponin I. To assess the performance of myosin heads in the presence of Glu150Ala mutant tropomyosin, we measured the polarized fluorescence of 1,5-IAEDANS probe bound to the SH1-helix of myosin. The obtained results indicate an abnormal increase in the number of myosin heads strongly bound to actin during relaxation of muscle fibres containing Glu150Ala mutant tropomyosin. It has been shown that the green tea catechin epigallocatechin gallate (EGCG), known as a modulator of troponin function, inhibits the premature transition of myosin heads into a state of strong actin binding, and thus weakens the damaging effect of the mutation. However, EGCG does not completely restore the effective behavior of myosin cross-bridges during the ATPase cycle.

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M. V. Tishkova

Institute of Cytology of the Russian Academy of Sciences

Author for correspondence.
Email: mariiatiskova@gmail.com
Russian Federation, Saint Petersburg, 194064

O. E. Karpicheva

Institute of Cytology of the Russian Academy of Sciences; Boston University

Email: mariiatiskova@gmail.com
Russian Federation, Saint Petersburg, 194064; Boston, 02118, MA, USA

Yu. S. Borovikov

Institute of Cytology of the Russian Academy of Sciences

Email: mariiatiskova@gmail.com
Russian Federation, Saint Petersburg, 194064

S. V. Avrova

Institute of Cytology of the Russian Academy of Sciences

Email: mariiatiskova@gmail.com
Russian Federation, Saint Petersburg, 194064

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2. Fig. 1. Control electrophoregram of Glu150Ala-mutant tropomyosin (Glu150Ala Tpm) binding to actin. Myosin and regulatory proteins were extracted from muscle fibers (track 1) to produce shadow fibers, the thin filaments of which were reconstructed by tropomyosin (track 3), and then by troponin. Isolated tropomyosin and actin are shown in tracks 2 and 4, respectively.

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3. Fig. 2. The effect of Glu150Ala substitution in tropomyosin on the orientation angle of the ΦE S1-AEDANS radiation dipoles in muscle fibers containing adjustable thin filaments in the absence and presence of the EGCG troponin inhibitor. Measurements were performed at low (10-8 M) and high (10-5 M) concentrations of Ca2 ions in the absence of nucleotides (+ Troponin) and in the presence of ADP and ATP. Changes in PH values between muscle fibers containing wild-type tropomyosin (WT-tropomyosin) and tropomyosin with Glu150Ala substitution (Glu150Ala-tropomyosin) (*), as well as between muscle fibers containing tropomyosin with Glu150Ala substitution in the absence and in the presence of EGCG (#) were significant at P < 0.05.

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4. Fig. 3. Effect of Glu150Ala substitution in tropomyosin on the number of disoriented 1.5-IAEDANS fluorophores associated with the myosin(N) SH1 helix in muscle fibers containing regulated thin filaments in the absence and presence of the EGCG troponin inhibitor. The measurements were performed at low (10-8 M) and high (10-5 M) concentrations of Ca2 ions in the absence of nucleotides (+ Troponin) and in the presence of ADP and ATP. Changes in N values between muscle fibers containing wild-type tropomyosin (WT-tropomyosin) and tropomyosin with Glu150Ala substitution (Glu150Ala-tropomyosin) (*), as well as between muscle fibers containing tropomyosin with Glu150Ala substitution in the absence and in the presence of EGCG (#) are significant at P < 0.05.

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