Phosphorylation of cardiac troponin I at protein kinase C site threonine 144 depresses cooperative activation of thin filaments

Abstract

There is evidence for PKC-dependent multisite phosphorylation of cardiac troponin I (cTnI) at Ser-23 and Ser-24 (also PKA sites) in the cardiac-specific N-terminal extension and at Thr-144, a unique residue in the inhibitory region. The functional effect of these phosphorylations in combination is of interest in view of data indicating intramolecular interaction between the N-terminal extension and the inhibitory region of cTnI. To determine the role of PKC-dependent phosphorylation of cTnI on sarcomeric function, we measured contractile regulation at multiple levels of complexity. Ca(2+) binding to thin filaments reconstituted with either cTnI(wild-type) or pseudo-phosphorylated cTnI(S23D/S24D), cTnI(T144E), and cTnI(S23D/S24D/T144E) was determined. Compared with controls regulated by cTnI(wild-type), thin filaments with cTnI(S23D/S24D) and cTnI(S23D/S24D/T144E) exhibited decreased Ca(2+) sensitivity. In contrast, there was no significant difference between Ca(2+) binding to thin filaments with cTnI(wild-type) and with cTnI(T144E). Studies of the pCa-force relations in skinned papillary fibers regulated by these forms of cTnI yielded similar results. However, in both the Ca(2+) binding measurements and the skinned fiber tension measurements, the presence of cTnI(S23D/S24D/T144E) induced a much lower Hill coefficient than either wild type, S23D/S24D, or T144E. These data highlight the importance of thin filament-based cooperative mechanisms in cardiac regulation, with implications for mechanisms of control of function in normal and pathological hearts.

FIGURE 1.

Determination of PKC-dependent cTnI phosphorylation by HPLC. A, SIM chromatogram of lysyl endopeptidase digests of PKCβ-treated cTn. Chromatogram of lysyl endopeptidase digest after 0 min (a, b) or 30 min (c, d) PKC-treated cTn are shown. B, relative abundance of phosphopeptides as a function of time after of PKC-treatment. Ser-23 and Ser-24 in cTnI peptide 1–36 and Thr-144 in cTnI peptide 141–164 were previously established to be the phosphorylated residues in this assay (8).

FIGURE 2.

Ca²⁺ binding to reconstituted thin filaments with recombinant cTnIs. A, relative fluorescence emission intensity changes of IAANS fluorescence attached to Cys-53 of cTnC. The reconstituted thin filaments contained either wild-type cTnI (circle) or pseudo-phosphorylated cTnI(T144E) (square), cTnI(S23D/S24D) (triangle), and cTnI(S23D/S24D/T144E) (diamond). B, reconstituted thin filaments contained either wild-type cTnI (circle), cTnI(T144A) (square), cTnI(S23A/S24A) (triangle), or cTnI(S23A/S24A/T144A) (diamond). C, reconstituted thin filaments contained either wild-type cTnI (circle), pseudo-phosphorylated cTnI(T144E) (square), PKA-treated wild-type cTnI (triangle), or PKA-treated cTnI(T144E) (diamond). S23D/S24D, as well as S23D/S24D/T144E, exhibited the expected decrease in Ca²⁺ binding affinity. However, S23D/S24D/T144E exclusively decreased the Hill coefficient of the Ca²⁺-dependent fluorescence change. Similar results were obtained from reconstituted thin filaments contained PKA-treated cTnI as shown in C and Table 1. Data are presented as mean ± S.E., n = 5–8.

FIGURE 3.

Effects of pseudo-phosphorylated cTnI on actomyosin S1-ATPase activity. The conditions were as follows: 35 mm NaCl, 5 mm MgCl₂, 20 mm MOPS, and either 2 mm EGTA or 0.1 mm CaCl₂, pH 7.0 at 25 °C. The concentrations of myosin S1, actin, and tropomyosin were 0.2, 5, and 2 μm respectively. The open and closed symbols indicate the presence of 2 mm EGTA and 0.1 mm CaCl₂, respectively. In the presence of EGTA, wild-type (circle), T144E (square), S23D/S24D (triangle), and S23D/S24D/T144E (diamond) all inhibited actomyosin S1-ATPase activity to a similar extent. In the presence of Ca²⁺, T144E, S23D/S24D, and S23D/S24D/T144E inhibited ATPase activity to varied degrees compared with wild type. Data are presented as mean ± S.E., n = 5–7. *, p < 0.05; **, p < 0.01 compared with wild type.

FIGURE 4.

ATPase activity in the absence and presence of Ca²⁺ with increasing concentrations of NEM-S1. The conditions were as follows: 35 mm NaCl, 5 mm MgCl₂, 20 mm MOPS, and either 2 mm EGTA or 0.1 mm CaCl₂, pH 7.0 at 25 °C. The concentrations of myosin S1, actin, tropomyosin, and troponin were 0.2, 5, 1, and 1 μm, respectively. Histograms display effect of cTnI on ATPase activity in the presence of EGTA (A) and in the presence of Ca²⁺ (B). Data are presented as mean ± S.E., n = 4–6.

FIGURE 5.

Effects of pseudo-phosphorylated cTnIs on force generation parameters of skinned fibers. A, force-pCa relationships in skinned cardiac muscle fibers into which wild-type cTnI (circle), or mutant of pseudo-phosphorylated cTnI(T144E) (square), cTnI(S23D/S24D) (triangle), or cTnI(S23D/S24D/T144E) (diamond) was incorporated. B, effects of the pseudo-phosphorylated cTnIs on the maximal force. The minimum and maximal force levels were not statistically significantly different between wild type and mutants. Data are presented as mean ± S.E., n = 4–5.