A functional and structural study of troponin C mutations related to hypertrophic cardiomyopathy

Abstract

Recently four new hypertrophic cardiomyopathy mutations in cardiac troponin C (cTnC) (A8V, C84Y, E134D, and D145E) were reported, and their effects on the Ca(2+) sensitivity of force development were evaluated (Landstrom, A. P., Parvatiyar, M. S., Pinto, J. R., Marquardt, M. L., Bos, J. M., Tester, D. J., Ommen, S. R., Potter, J. D., and Ackerman, M. J. (2008) J. Mol. Cell. Cardiol. 45, 281-288). We performed actomyosin ATPase and spectroscopic solution studies to investigate the molecular properties of these mutations. Actomyosin ATPase activity was measured as a function of [Ca(2+)] utilizing reconstituted thin filaments (TFs) with 50% mutant and 50% wild type (WT) and 100% mutant cardiac troponin (cTn) complexes: A8V, C84Y, and D145E increased the Ca(2+) sensitivity with only A8V demonstrating lowered Ca(2+) sensitization at the 50% ratio when compared with 100%; E134D was the same as WT at both ratios. Of these four mutants, only D145E showed increased ATPase activation in the presence of Ca(2+). None of the mutants affected ATPase inhibition or the binding of cTn to the TF measured by co-sedimentation. Only D145E increased the Ca(2+) affinity of site II measured by 2-(4'-(2''-iodoacetamido)phenyl)aminonaphthalene-6-sulfonic acid fluorescence in isolated cTnC or the cTn complex. In the presence of the TF, only A8V was further sensitized to Ca(2+). Circular dichroism measurements in different metal-bound states of the isolated cTnCs showed changes in the secondary structure of A8V, C84Y, and D145E, whereas E134D was the same as WT. PyMol modeling of each cTnC mutant within the cTn complex revealed potential for local changes in the tertiary structure of A8V, C84Y, and D145E. Our results indicate that 1) three of the hypertrophic cardiomyopathy cTnC mutants increased the Ca(2+) sensitivity of the myofilament; 2) the effects of the mutations on the Ca(2+) affinity of isolated cTnC, cTn, and TF are not sufficient to explain the large Ca(2+) sensitivity changes seen in reconstituted and fiber assays; and 3) changes in the secondary structure of the cTnC mutants may contribute to modified protein-protein interactions along the sarcomere lattice disrupting the coupling between the cross-bridge and Ca(2+) binding to cTnC.

FIGURE 1.

The actin-Tm-activated myosin ATPase activity of HCM cTnC mutants as a function of pCa. A, HcTnC mutant or WT at 100%. B, HcTnC mutants and WT at a 50% to 50% ratio. The experiments were performed using 0.6 μm myosin, 3.5 μm actin, 1 μm Tm, and 1 μm preformed Tn complex. The buffer conditions were as described under “Experimental Procedures.” The basal ATPase activity at pCa 8.0 was considered 0%. The intermediate points were normalized to the maximal ATPase activity at pCa 4.0 and were considered 100%. The specific ATPase activity at pCa 8.0 was 0.15, 0.19, 0.11, 0.19, and 0.18 mol of P_i × mol of myosin⁻¹ × s⁻¹ for WT, A8V, C84Y, E134D, and D145E, respectively. Each curve represents an average of six to seven experiments, and error is reported as mean ± S.E.

FIGURE 2.

The activation and inhibition of the actin-Tm-activated myosin ATPase activity by different HCM cTnC mutants in the presence and absence of Ca²⁺. A, activation of actomyosin ATPase at 1 μm Tn complex in the presence of Ca²⁺. B, inhibition of preformed actomyosin ATPase activity by HCM cTnC mutants at increasing ratios of Tn complex as indicated on the abscissa. The assay conditions were as follows: 3.5 μm actin, 1 μm Tm, and 0.6 μm myosin were dissolved in 75 mm KCl, 3.3 mm MgCl₂, 1.7 mm CaCl₂ (for activation assay) or 0.13 μm CaCl₂ (for inhibition assay), 1.5 mm EGTA, 3.5 mm ATP, 1 mm DTT, 11.5 mm MOPS, pH 7.0. The myosin ATPase activity that occurs in the absence of cTn complex is considered 100% ATPase activity. The specific ATPase activity in the absence of troponin complexes was measured as 0.35 mol of P_i × mol of myosin⁻¹ × s⁻¹. Each point represents an average of six experiments performed in triplicate and is expressed as mean ± S.E. *, p < 0.05 compared with WT.

FIGURE 3.

Co-sedimentation of actin, tropomyosin, and Tn complexes containing HCM cTnC mutants. The experimental design and buffer conditions were as described under “Experimental Procedures.” Samples were separated by 15% SDS-PAGE. S, supernatant; P, pellet.

FIGURE 4.

Determination of apparent Ca²⁺ affinities of isolated HCM cTnC mutants by fluorescence. Steady state fluorescence measurements excited the IAANS probe at 330 nm, and emission was detected at 450 nm. A, HcTnCs doubly labeled at cysteines 35 and 84: WT^IAANS35^IAANS84, A8V^IAANS35^IAANS84, E134D^IAANS35^IAANS84, and D145E^IAANS35^IAANS84. B, HcTnCs labeled at cysteine 35: C84S^IAANS35 (control) and C84Y^IAANS35. Fluorescence spectral changes were recorded during the titration of microliter amounts of CaCl₂ to a 2-ml experimental volume. The concentration of free Ca²⁺ and amounts of titrated Ca²⁺ were calculated using the pCa calculator program developed by our laboratory (see “Experimental Procedures”). The program made corrections for dilution effects that occur during titration of Ca²⁺. The data were fitted to a version of the Hill equation that accounted for the spectral changes occurring at low Ca²⁺ concentration. The Ca²⁺ affinities are reported in Table 1 as pCa₅₀ and n_Hill values ±S.E. Each point represents an average of four to five experiments and is expressed as mean ± S.E.

FIGURE 5.

Determination of apparent Ca²⁺ affinities of cTn and thin filament HCM mutants by fluorescence. Steady state fluorescence measurements excited the IAANS probe at 330 nm, and emission was detected at 450 nm. A, cTn complexes containing HCM HcTnC mutants that were doubly labeled at cysteines 35 and 84. B, TF-containing HCM HcTnC mutants that were singly labeled at cysteine 35. The WT, A8V, E134D, and D145E had the cysteine 84 mutated allowing labeling at a single site. Fluorescence spectral changes were recorded during the titration of microliter amounts of CaCl₂ to a 2-ml experimental volume. The data were fitted to a version of the Hill equation that accounted for the spectral changes occurring at low Ca²⁺ concentration. The Ca²⁺ affinities are reported in Table 2 as pCa₅₀ values ±S.E. Each point represents an average of four to five experiments and is expressed as mean ± S.E.

FIGURE 6.

Circular dichroism spectra of wild type and HCM cTnC mutations. Far-UV CD spectra were performed in apo, Mg²⁺-bound, and Ca²⁺/Mg²⁺-bound states. Spectra were recorded at 195–250 nm utilizing a 1-mm-path quartz cell in a Jasco-720 spectropolarimeter at room temperature (20 °C). The spectra shown are the average of three independent measurements. For each independent measurement 10 scans were averaged, and no numerical smoothing was applied. The optical activity of the buffer was subtracted from relevant protein spectra. The buffer conditions were as described under “Experimental Procedures.” Mean residue ellipticity ([θ]_MRE in degree·cm²/dmol) for the spectra was calculated utilizing the same Jasco system software and the following equation: [θ]_MRE = [θ]/(10 × Cr × l) where [θ] is the measured ellipticity in millidegrees, Cr is the mean residue molar concentration, and l is the path length in cm. deg, degrees.

FIGURE 7.

Mapping and modeling of D145E HCM mutation in HcTnC in Mg²⁺-loaded C terminus and Ca²⁺-saturated states. cTnC HCM mutation D145E modeled into the NMR structure of the Mg²⁺-loaded C terminus of cTnC-(81–161) complexed with cTnI-(33–80) (Protein Data Bank code 1SBJ (63)) and Ca²⁺-saturated cTn complex crystal structure (Protein Data Bank (PDB) code 1J1E (21)) is shown. A, location of the residue Asp-145 in the Mg²⁺-bound state and H-bonding shown with red dotted lines. B, E145 modeled into Protein Data Bank structure 1SBJ showing H-bonding contacts. C, the residue Asp-145 shown in the Ca²⁺-bound cTn structure (Protein Data Bank code 1J1E). Helices in cTnC are indicated, and the interacting portion of cTnT is labeled. D, the mutation D145E modeled into the Ca²⁺-bound structure (Protein Data Bank code 1J1E) showing residues that make contact through H-bonding. E, different orientation of modeled mutation D145E.