Recent studies of the molecular mechanism of lusitropy due to phosphorylation of cardiac troponin I by protein kinase A

Figures

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  Fig. 1. The effects of β1 receptor activation on cardiac myocyte contractility. Rat myocytes paced at 1 Hz, 37 °C. 1 µM dobutamine increases shortening amplitude and reduces contraction time (TTP90) and relaxation time (TTB90). Colours represent fitted parameters as described in the figure. Plots on the right show dose–response curves. Data from Wright et al. (2020)

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  Fig. 2. Evolutionary timeline for the development of the N-terminal phosphorylatable peptide of cTnI and lusitropy. Figure kindly provided by Dr J-P Lin, Univ Illinois

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  Fig. 3. Small molecules that can restore the response to phosphorylation in mutant thin filaments. Measurement made by in vitro motility assay in reconstituted human thin filaments. Left: fixed [Ca2+] assay (~ ec50) using WT or TPM1 E180G HCM mutant. Small molecules do not affect wild-type but EGCG, Silybin B and resveratrol; restore the phosphorylation effect whilst Silybin A does not. The same effect of the small molecules is shown by the measurements of the Ca2+-activation curve for phosphorylated and unphosphorylated DCM mutant (TNNC1 G159D) thin filaments

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  Fig. 4. Structural representation of the backbone for the most populated state of the unphosphorylated troponin core, determined by molecular dynamics simulation. The peptide chains are coloured according to their RMSF, with the highest RMSF deepest red. The most mobile areas are NcTnI (1–32), CcTnT (278–288) and cTnI “inhibitory” peptide (137–148). This result is very similar to that measured by H–D Exchange (Kowlessur and Tobacman, 2012). Serines 22 and 23 represented as spheres. TnC is coloured green

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  Fig. 5. cTnC backbone 15 N R2 relaxation time measurements for TnC-TnI peptide complexes. Plots of 15 N R2 relaxation time vs cTnC residue number for free cTnC and complexes with cTnI 1–77 Left: comparison of 15 N R2 relaxation rates of free cTnC, cTnC complexed with wildtype cTnI1-77, and cTnC complexed with phosphomimetic cTnI1-77 S22/23D. Right: Comparison of 15 N R2 relaxation rates of free cTnC, cTnC complexed with wildtype cTnI1-77, and substituted DCM mutant cTnC G159D. Figure kindly supplied by Dr P Hwang, Univ Alberta

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  Fig. 6. Wild Type molecular dynamics representative structure showing NcTnC and NcTnI only in unphosphorylated and phosphorylated states. The major phosphorylation-dependent interactions between NcTnC and NcTnI side chains are shown. NcTnI (1–34) is shown in blue, NcTnC is shown in brown (unphosphorylated) and pale blue (phosphorylated). Helix A and B are indicated. Figure kindly supplied by Zeyu Yang

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  Fig. 7. Comparison of the effect of phosphorylation of cardiac troponin on its biochemical, physiological and molecular dynamics parameters, its suppression by the TnC G159D DCM related mutation and its restoration by small molecules. The response to phosphorylation measured by an in vitro motility screen at fixed Ca2+ (data from Sheehan et al. 2018). Measurement of the change in thin filament Ca.2+ sensitivity on phosphorylation expressed a EC50 uP/P ratio, measured by motility assay (data from Sheehan et al. 2018). Lusitropic effect measured in wild-type or mutant cardiomyocytes paced at 1 Hz, 37 °C expressed as the fractional change in ttb90 due to dobutamine (see Fig. 1; data from Sheehan, 2019). Change in the A/B and interdomain hinge angles on phosphorylation calculated by molecular dynamics simulations are shown (data from Yang et al 2021). SA, SilybinA; SB, SilybinB; EGCG, epigallocatechin-3 gallate. DCM mutations block the response to phosphorylation but it can be restored by Silybin B, EGCG and Resveratrol (Yang et al. 2021)

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  Fig. 8. Position of the cardiac N-terminal TnI domain in the thin filament model. (Left) Initial model showing the troponin core-domain and the N-terminal extension of cardiac TnI abutting actin and tropomyosin (one actin subunit shown, grey; tropomyosin, magenta/purple; TnC, orange; TnT, yellow; TnI residues 41–171, cyan; and N-terminal TnI residues 1–40, blue with its Ser23/24 residues coloured green). (middle) Surface rendering of the central segment of the Yamada et al. cryo-EM reconstruction of cardiac thin filaments (right) The thin filament model superimposed on the cryo-EM thin filament volume in made translucent. Figure kindly provided by Dr W Lehman, Boston University