Phosphorylation of cMyBP-C affects contractile mechanisms in a site-specific manner

Abstract

Cardiac myosin binding protein-C (cMyBP-C) is a cardiac-specific, thick-filament regulatory protein that is differentially phosphorylated at Ser(273), Ser(282), and Ser(302) by various kinases and modulates contraction. In this study, phosphorylation-site-specific effects of cMyBP-C on myocardial contractility and cross-bridge kinetics were studied by sinusoidal analysis in papillary and trabecular muscle fibers isolated from t/t (cMyBP-C-null) mice and in their counterparts in which cMyBP-C contains the ADA (Ala(273)-Asp(282)-Ala(302)), DAD (Asp(273)-Ala(282)-Asp(302)), and SAS (Ser(273)-Ala(282)-Ser(302)) mutations; the results were compared to those from mice expressing the wild-type (WT) transgene on the t/t background. Under standard activating conditions, DAD fibers showed significant decreases in tension (~50%), stiffness, the fast apparent rate constant 2πc, and its magnitude C, as well as its magnitude H, but an increase in the medium rate constant 2πb, with respect to WT. The t/t fibers showed a smaller drop in stiffness and a significant decrease in 2πc that can be explained by isoform shift of myosin heavy chain. In the pCa-tension study using the 8 mM phosphate (Pi) solution, there was hardly any difference in Ca(2+) sensitivity (pCa50) and cooperativity (nH) between the mutant and WT samples. However, in the solutions without Pi, DAD showed increased nH and slightly decreased pCa50. We infer from these observations that the nonphosphorylatable residue 282 combined with phosphomimetic residues Asp(273) and/or Asp(302) (in DAD) is detrimental to cardiomyocytes by lowering isometric tension and altering cross-bridge kinetics with decreased 2πc and increased 2πb. In contrast, a single change of residue 282 to nonphosphorylatable Ala (SAS), or to phosphomimetic Asps together with the changes of residues 273 and 302 to nonphosphorylatable Ala (ADA) causes minute changes in fiber mechanics.

Figure 1

Slow time courses of length (A) and tension (B), demonstrating the experimental protocol. This example is from an ADA fiber preparation (0.9 mm in length and 87 μm in diameter). The amplitude of sinusoidal oscillation was kept at 0.125% L₀ in all frequencies, but because the signal was filtered with a second-order low-pass filter (cutoff at 10 Hz), the amplitude looks attenuated at higher frequencies (to the left). R, relaxing solution; A, standard activating solution. ^∗2 indicates that the solution was changed twice.

Figure 2

Isometric tension (T_max in Eq. 2; left ordinate) and stiffness (S_max in Eq. 3; right ordinate) in 8Pi solution (A) and 0Pi solution (B) at saturating [Ca²⁺]. The 8Pi solution contained 8 mM Pi and its IS was 200 mM, whereas the 0Pi solution contained no added Pi and its IS was 150 mM. ^∗∗p < 0.01 and ^∗p < 0.05 compared to WT. The number of experiments (n) in A is 31 for WT, 38 for t/t, 32 for ADA, 31 for DAD, and 27 for SAS; in B, n = 14 for WT, t/t, ADA, and DAD, and n = 11 for SAS.

Figure 3

Averaged complex modulus data in the 8Pi solution. Data were plotted as elastic modulus (real Y(f)) versus frequency (f) (A), viscous modulus (imaginary Y(f)) versus frequency (B), and viscous modulus versus elastic modulus (Nyquist plot) (C). n = 31 for WT, 38 for t/t, 32 for ADA, 31 for DAD, and 27 for SAS. Solid lines represent Eq. 1 with best-fit parameters. In C, frequency increases in the clockwise direction; its value can be found in A and B.

Figure 4

Parameters of exponential processes in the 8Pi solution (pCa 4.55). (A) Apparent rate constants 2πb (left ordinate) and 2πc (right ordinate). (B) Magnitudes of processes B (left ordinate) and C (right ordinate). (C) Magnitude H. Error bars represent the mean ± SE of 26–38 experiments carried out in each group (for numbers of mice, see Table 1). n = 31 for WT, 38 for t/t, 32 for ADA, 31 for DAD, and 27 for SAS. ^∗∗p < 0.01 and ^∗p < 0.05 compared to WT.

Figure 5

Parameters of exponential processes in the 0Pi solution (pCa 4.40). (A) Apparent rate constants 2πb (left ordinate) and 2πc (right ordinate). (B) Magnitudes of processes B (left ordinate) and C (right ordinate). (C) Magnitude H. n = 14 for WT, t/t, ADA, and DAD, and n = 11 for SAS. ^∗∗p < 0.01 and ^∗p < 0.05 compared to WT.

Figure 6

Effect of pCa in the 8Pi solution in five groups of mice. (A) pCa-tension curves normalized to T_max. (B) pCa₅₀ (left ordinate) and n_H (right ordinate), as deduced by fitting the individual data to Eq. 2, and averaging. (C) pCa-stiffness curves normalized to S_max. (D) pCa_50S (left ordinate) and n_HS (right ordinate), deduced by fitting the data to Eq. 3, and averaging. In A and C, the data are shown by discrete points, and the best-fit curves are shown by solid lines. B and D do not reveal significant differences in any of the parameters measured. n = 17 for WT, 13 for t/t, 14 for ADA, 18 for DAD, and 12 for SAS.

Figure 7

Effect of pCa in the 0Pi solution in five groups of mice. Nomenclatures and conventions used are as in Fig. 6. n = 14 for WT, t/t, ADA, and DAD, and n = 11 for SAS. ^∗∗p ≤ 0.01, ^∗0.01 < p ≤ 0.05, and (^∗)0.05 < p ≤ 0.1 compared to WT.

Figure 8

Apparent rate constants 2πb (A) and 2πc (B) plotted against pCa in the 8Pi solution. Values at pCa ≥ 6.0 are not included, because they were seriously contaminated with noise. n = 17 for WT, 13 for t/t, 14 for ADA, 18 for DAD, and 12 for SAS.

Figure 9

Expression of myosin α- and β-isoforms in each mouse group. (A) A representative experiment. Isoforms detected using specific antibodies after Sypro stained SDS-PAGE analysis. The molecular mass of α-MHC is 223.565 kDa, and that of β-MHC is 222.849 kDa. (B) Fractions of α- and β-MHC, as averaged from three gels (n = 3), similar to that represented in A, for WT and mutant cMyBP-C. Error bars are drawn for each fraction. ^∗p ≤ 0.05.