PKCα-Specific Phosphorylation of the Troponin Complex in Human Myocardium: A Functional and Proteomics Analysis

<div><p>Aims</p><p>Protein kinase Cα (PKCα) is one of the predominant PKC isoforms that phosphorylate cardiac troponin. PKCα is implicated in heart failure and serves as a potential therapeutic target, however, the exact consequences for contractile function in human myocardium are unclear. This study aimed to investigate the effects of PKCα phosphorylation of cardiac troponin (cTn) on myofilament function in human failing cardiomyocytes and to resolve the potential targets involved.</p><p>Methods and Results</p><p>Endogenous cTn from permeabilized cardiomyocytes from patients with end-stage idiopathic dilated cardiomyopathy was exchanged (∼69%) with PKCα-treated recombinant human cTn (cTn (DD+PKCα)). This complex has Ser23/24 on cTnI mutated into aspartic acids (D) to rule out <i>in vitro</i> cross-phosphorylation of the PKA sites by PKCα. Isometric force was measured at various [Ca²⁺] after exchange. The maximal force (F_max) in the cTn (DD+PKCα) group (17.1±1.9 kN/m²) was significantly reduced compared to the cTn (DD) group (26.1±1.9 kN/m²). Exchange of endogenous cTn with cTn (DD+PKCα) increased Ca²⁺-sensitivity of force (pCa₅₀ = 5.59±0.02) compared to cTn (DD) (pCa₅₀ = 5.51±0.02). In contrast, subsequent PKCα treatment of the cells exchanged with cTn (DD+PKCα) reduced pCa₅₀ to 5.45±0.02. Two PKCα-phosphorylated residues were identified with mass spectrometry: Ser198 on cTnI and Ser179 on cTnT, although phosphorylation of Ser198 is very low. Using mass spectrometry based-multiple reaction monitoring, the extent of phosphorylation of the cTnI sites was quantified before and after treatment with PKCα and showed the highest phosphorylation increase on Thr143.</p><p>Conclusion</p><p>PKCα-mediated phosphorylation of the cTn complex decreases F_max and increases myofilament Ca²⁺-sensitivity, while subsequent treatment with PKCα in situ decreased myofilament Ca²⁺-sensitivity. The known PKC sites as well as two sites which have not been previously linked to PKCα are phosphorylated in human cTn complex treated with PKCα with a high degree of specificity for Thr143.</p></div

Effects of cTn (DD) and cTn (DD+PKCα) exchange in failing cardiomyocytes.

<p>(<b>A</b>) The maximal force generating capacity of the cardiomyocytes was significantly reduced in the cells exchanged with cTn (DD+PKCα) compared to the cTn (DD) group and the cTn (DD) group. *P<0.05, <i>t-</i>test. The passive force, as well as the steepness of the force–pCa relation (nH), and the rate of force redevelopment (K_tr-max) were not significantly different among the groups (<b>B–D</b>). The maximum force at saturating Ca²⁺-concentration was significantly reduced in the cardiomyocytes exchanged with cTn (DD+PKCα). The combined effects of cTn exchange on maximal force and Ca²⁺-sensitivity resulted in a depressed force in the cTn (DD+PKCα) group compared to the cTn (DD) group at maximal and submaximal Ca²⁺ concentrations (* P<0.05 in <i>t</i>-test) (<b>E</b>).</p

Patient characteristics.

<p>LVEF, LV ejection fraction; F, Female; M, Male. Medication used included angiotensin-converting-enzyme inhibitors, angiotensin II receptor antagonists, β-blockers, digoxin and anti-arrhythmic agents.</p

Novel identified PKCαphosphorylation sites on cTnI and cTnT: Ser198 on cTnI and Ser179 on cTnT identified in human recombinant cTn complex.

<p>(<b>A</b>) MS/MS spectrum of the m/z 581.78 unphosphorylated peptide (<b>B</b>) MS/MS spectrum of the m/z 621.76 phosphorylated peptide. (<b>C</b>) MS/MS spectrum of the m/z 799.39 unphosphorylated peptide (<b>D</b>) MS/MS spectrum of the m/z 838.87 phosphorylated peptide. The b-ions denote N-terminal ions and y-ions denote C-terminal ions. The tandem MS (MS/MS) of our phosphorylated peptide ions undergo a preferential neutral loss of H₃PO₄ (98 Da). The MS/MS patterns confirm the phosphorylation of the two peptides and identify the novel phosphorylation sites.</p

PKCα-mediated phosphorylation of human recombinant cTn complex (cTn (DD)).

<p>(<b>A</b>) Samples were taken at different time points and run on a 1D gradient gel stained with ProQ Diamond and with Sypro Ruby (M: Peppermint molecular weight marker). (<b>B</b>) Time course of PKCα-mediated phosphorylation of recombinant cTn (DD) complex. Mean values are shown (±S.E.M.) obtained from two different 1D-gels; the intensities of the cTnI bands on the Sypro Ruby stained gels were used to correct for loading differences. Time constant (±S.E.M.) of the exponential fitted to the data points: 60±3 minutes for cTnT and 58±4 minutes for cTnI phosphorylation. (<b>C</b>) Western blotting of the cTn (DD) complex incubated with PKCα for 90 minutes (cTn (DD+PKCα)) showed phosphorylation at cTnI sites Ser42 and Thr143 with phospho-specific antibodies.</p

Effect of cTn (DD+PKCα) exchange on the Ca²⁺-sensitivity of force.

<p>(<b>A</b>) The Ca²⁺-sensitivity derived from the midpoint of the force-pCa relationship (pCa₅₀) increased upon exchange with cTn (DD+PKCα) compared to failing cardiomyocytes exchanged with cTn (DD). *P<0.05, cTn (DD) exchange vs. cTn (DD+PKCα) exchange in ANOVA, followed by Bonferroni post-hoc test. Time control denotes results obtained from cardiomyocytes kept in exchange buffer without cTn complex added. (<b>B</b>) Subsequent incubation of PKCα of the cardiomyocytes exchanged with cTn (DD+PKCα) significantly reduced Ca²⁺-sensitivity of force development. *P<0.05 cTn (DD+PKCα) vs. cTn (DD+PKCα) + PKCα in paired <i>t</i>-test.</p

Essentiality of <i>eccC</i>_<i>5</i> and analysis of functional domains.

<p>* The <i>eccC</i>_<i>5</i> NBD mutants appear to have a dominant negative effect on the functioning of endogenous EccC₅.</p><p>^$ Colonies showed a strong growth defect, i.e. colonies were visible only after 17 days, compared to 10 days for the wild-type strain.</p><p>Replacement of pMV-<i>eccBC</i>_<i>5</i> by the input DNA was scored. Input DNA consisted of the pMV-361-<i>hyg</i> plasmid containing the indicated constructs. “+” indicates that more than 100 colonies were detected after electroporation with the indicated vector. “–” indicates between 0–20 colonies were found after electroporation. These latter colonies were shown by PCR to still contain the original vector, indicating illegitimate recombination or spontaneous antibiotic resistance. Results are representative data of three independent experiments.</p><p>Essentiality of <i>eccC</i>_<i>5</i> and analysis of functional domains.</p

Complementation of <i>mas</i>::<i>tn</i> in the Δ<i>eccC</i>_<i>5</i> mutant by replacement of the integrated pMV vector.

<p>Indicated strains were electroporated with the input DNA shown on the left. Input DNA consisted of the pMV-361-<i>hyg</i> plasmid containing the indicated constructs. Valid insertion of input DNA was scored as + or-, similarly as described for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005190#pgen.1005190.t001" target="_blank">Table 1</a>. Results are representative data of three independent experiments.</p><p>Complementation of <i>mas</i>::<i>tn</i> in the Δ<i>eccC</i>_<i>5</i> mutant by replacement of the integrated pMV vector.</p

Site-specific quantification of PKCα-treated human donor and failing tissue and recombinant cTnI using MRM. A

<p>. The fold increase of phosphorylation of Ser198 on cTnI in Control (untreated tissue) and PKCα-treated donor and failing tissue (<i>n</i> = 4 per group; technical replicates  = 3). All values from the tissue samples were determined using synthesized internal standard peptides, and expressed relative to total cTnI content. Error bars indicate the standard error of mean (SEM). * <i>P</i><0.005 in unpaired student <i>t</i>-test <b>B</b>. To quantify the phosphorylation status of each site in recombinant cTnI incubated with PKCα, MRM assays were designed for each mono or di-phosphorylated sequence and analyzed (technical replicates  = 4). The obtained values were corrected for loading differences by the intensity of the coomassie-stained excised gel bands. A significant increased phosphorylation was observed for all the phosphorylation sites after PKCα-treatment. A zoom-in of the Ser198 bar has been inserted on the side for clarification. *P<0.05, time-point <1 minute vs. time-point 180 minutes in paired <i>t</i>-test.</p

Overview of cardiomyocyte force measurements.

<p>Time control denotes results obtained from cardiomyocytes kept in exchange buffer without cTn complex added. Symbols used: # denotes P<0.05, cTn (DD+PKCα) compared to time control or cTn (DD) and † denotes P<0.05, cTn (DD) compared to time control in ANOVA followed by Bonferroni post-hoc test. Measurements in cTn exchanged cells were repeated after treatment with PKCα. In this case * denotes P<0.05, before vs. after incubation with PKCα in paired <i>t</i>-test. Abbreviations: F_max, maximal force per cross-sectional area at saturating calcium concen-tration (pCa4.5) in kN/m²; F_pas, passive force per cross-sectional area in relaxing solution (pCa 9) in kN/m²; nH, steepness of the force-pCa curves; K_tr-max, the rate of force redevelopment at maximal activating solution (pCa 4.5) in s⁻¹.</p