cTn Complex Research Articles

Effect of cTnC Mutations on the Response of Reconstituted Thin Filaments to Calcium Upon cTnI Phosphorylation

The objective of this work is to examine the effect of calcium sensitizing and desensitizing cTnC mutations on the response of reconstituted thin filaments to calcium upon cTnI phosphorylation. M45Q cTnC mutation increases calcium sensitivity of the cTn complex reconstituted into thin filaments by decreasing the rate of calcium dissociation. On the other hand, the D73N mutation (removal of an X-acid pair from the second EF-hand of cTnC) reduces the calcium sensitivity of the cTn complex reconstituted into thin filaments by accelerating the rate of calcium dissociation. Pseudo-phosphorylation of cTnI by PKA led to ∼2.8-fold decrease in the calcium sensitivity of the cTn complex reconstituted into thin filaments. In addition, pseudo-phosphorylation of cTnI led to ∼3.3-fold faster rate of calcium dissociation from the cTn complex reconstituted into thin filaments. However, the D73N mutation substantially diminished the extent of the decrease in calcium sensitivity and of the acceleration in calcium dissociation from the cTn complex reconstituted into thin filaments associated with pseudo-phosphorylation of cTnI by PKA. In contrast, M45Q mutation did not abolish the decrease in the calcium sensitivity and the acceleration in calcium dissociation from the cTn complex reconstituted into thin filaments induced by the pseudo-phosphorylation of cTnI by PKA. In fact, M45Q mutation enhanced the effect of cTnI pseudo-phosphorylation on the rate of Ca2+ dissociation from the cTn complex reconstituted into thin filaments. These results suggest that calcium desensitizing cTnC mutations diminish the effect of phosphorylation of cTnI on calcium binding and exchange with reconstituted thin filaments. A deeper understanding of how altered calcium sensitivity of cTnC affects the transmission of PKA phosphorylation signal from cTnI to cTnC could lead to the rational design of novel treatments for heart disease.

279 Right ventricular metabolic imaging in experimental pulmonary artery hypertension

fore, wild type and mutant L29Q cTn were reconstituted with the pseudo-phosphorylated S22D/S23D cTnI that mimics the native phosphorylated state to determine the mechanism of FHC and its effect on cardiac function. METHODS/RESULTS: To monitor the differences of the calcium binding affinity for the wild type and the mutant cTn, a novel cTnC construct (cTnC) labelled with IAANS molecule was used. Upon a series of calcium titrations, the IAANS-fluorescence changes induced by different cTn complexes were measure by a Cary Eclipse spectrophotometer. Our current results indicate that the L29Q mutation increases the calcium binding affinity of cTn, consistent with previous findings. However, contrary to previous findings, the pseudo-phosphorylated cTnI appears to increase calcium binding affinity for the wild type cTn. In addition, this increased of calcium binding affinity caused by the pseudo-phosphorylated cTnI is not observed in L29Q cTn, suggesting that the interaction between cTnC and cTnI may be disrupted by the L29Q mutation. CONCLUSION: Our fluorescence study shows that the reconstituted L29Q cTn has an overall higher calcium binding affinity than that of the wild type cTn. In addition, the presence of the pseudo-phosphorylated cTnI increases the calcium affinity only for the wild type cTn but not for the L29Q cTn. This suggests that L29Q mutation may potentially disrupt the interaction between cTnC and cTnI and thus leads to the impaired cardiac relaxation pertain to the etiology of FHC. Heart and Stroke Foundation of BC & Yukon

The influence of trout cardiac troponin I and PKA phosphorylation on the Ca2+ affinity of the cardiac troponin complex

The trout heart is 10-fold more sensitive to Ca(2+) than the mammalian heart. This difference is due, in part, to cardiac troponin C (cTnC) from trout having a greater Ca(2+) affinity than human cTnC. To determine what other proteins are involved, we cloned cardiac troponin I (cTnI) from the trout heart and determined how it alters the Ca(2+) affinity of a cTn complex containing all mammalian components (mammalian cTn). Ca(2+) activation of the complex was characterized using a human cTnC mutant that contains anilinonapthalenesulfote iodoacetamide attached to Cys53. When the cTn complex containing labeled human cTnC was titrated with Ca(2+), its fluorescence changed, reaching an asymptote upon saturation. Our results reveal that trout cTnI lacks the N-terminal extension found in cTnI from all other vertebrate groups. This protein domain contains two targets (Ser23 and Ser24) for protein kinase A (PKA) and protein kinase C. When these are phosphorylated, the rate of cardiomyocyte relaxation increases. When rat cTnI in the mammalian cTn complex was replaced with trout cTnI, the Ca(2+) affinity was increased ∼1.8-fold. This suggests that trout cTnI contributes to the high Ca(2+) sensitivity of the trout heart. Treatment of the two cTn complexes with PKA decreased the Ca(2+) affinity of both complexes. However, the change for the complex containing rat cTnI was 2.2-fold that of the complex containing trout cTnI. This suggests that the phosphorylation of trout cTnI does not play as significant a role in regulating cTn function in trout.

Cardiac Troponin T: A Sarcomeric AKAP, Tethers Protein Kinase a at the Myofilaments

Efficient and specific phosphorylation of PKA substrates, elicited in response to beta-adrenergic stimulation, require spatially confined pools of PKA anchored in proximity of its substrates. PKA-dependent phosphorylation of cardiac sarcomeric proteins has been the subject of intense investigations. Yet, the identity, composition, and function of PKA complexes at the sarcomeres have remained elusive. Here we report the identification and characterization of a novel sarcomeric AKAP (A kinase anchoring protein), cardiac troponin T (cTnT). Using the yeast two-hybrid technology in screening two adult human heart cDNA libraries, we identified the regulatory subunit of PKA as interacting with human cTnT bait. Immunoprecipitation studies show that cTnT is a dual specificity AKAP, interacting with both PKA-regulatory subunits type I and II. The disruptor peptide Ht31, but not Ht31P (control), abolished cTnT/PKA-R association. Truncations and point mutations identified an amphipathic helix domain in cTnT as the PKA binding site. This was confirmed by a peptide SPOT assay in the presence of Ht31 or Ht31P (control). Gelsolin-dependent removal of thin filament proteins also reduced myofilament-bound PKA-type II. Using a cardiac troponin (cTn) exchange procedure that substitutes the endogenous cTn complex with a recombinant cTn complex we show PKA-type II is troponin-bound in the myofilament lattice. Displacement of PKA-cTnT complexes correlates with a significant decrease in myofibrillar PKA activity. Taken together, our data propose a novel role for cTnT as a dual-specificity sarcomeric AKAP.

Cardiac Troponin T, a Sarcomeric AKAP, Tethers Protein Kinase A at the Myofilaments

Efficient and specific phosphorylation of PKA substrates, elicited in response to β-adrenergic stimulation, require spatially confined pools of PKA anchored in proximity of its substrates. PKA-dependent phosphorylation of cardiac sarcomeric proteins has been the subject of intense investigations. Yet, the identity, composition, and function of PKA complexes at the sarcomeres have remained elusive. Here we report the identification and characterization of a novel sarcomeric AKAP (A-kinase anchoring protein), cardiac troponin T (cTnT). Using yeast two-hybrid technology in screening two adult human heart cDNA libraries, we identified the regulatory subunit of PKA as interacting with human cTnT bait. Immunoprecipitation studies show that cTnT is a dual specificity AKAP, interacting with both PKA-regulatory subunits type I and II. The disruptor peptide Ht31, but not Ht31P (control), abolished cTnT/PKA-R association. Truncations and point mutations identified an amphipathic helix domain in cTnT as the PKA binding site. This was confirmed by a peptide SPOT assay in the presence of Ht31 or Ht31P (control). Gelsolin-dependent removal of thin filament proteins also reduced myofilament-bound PKA-type II. Using a cTn exchange procedure that substitutes the endogenous cTn complex with a recombinant cTn complex we show that PKA-type II is troponin-bound in the myofilament lattice. Displacement of PKA-cTnT complexes correlates with a significant decrease in myofibrillar PKA activity. Taken together, our data propose a novel role for cTnT as a dual-specificity sarcomeric AKAP.

PLEKHA7 Is an Adherens Junction Protein with a Tissue Distribution and Subcellular Localization Distinct from ZO-1 and E-Cadherin

The pleckstrin-homology-domain-containing protein PLEKHA7 was recently identified as a protein linking the E-cadherin-p120 ctn complex to the microtubule cytoskeleton. Here we characterize the expression, tissue distribution and subcellular localization of PLEKHA7 by immunoblotting, immunofluorescence microscopy, immunoelectron microscopy, and northern blotting in mammalian tissues. Anti-PLEKHA7 antibodies label the junctional regions of cultured kidney epithelial cells by immunofluorescence microscopy, and major polypeptides of Mr ∼135 kDa and ∼145 kDa by immunoblotting of lysates of cells and tissues. Two PLEKHA7 transcripts (∼5.5 kb and ∼6.5 kb) are detected in epithelial tissues. PLEKHA7 is detected at epithelial junctions in sections of kidney, liver, pancreas, intestine, retina, and cornea, and its tissue distribution and subcellular localization are distinct from ZO-1. For example, PLEKHA7 is not detected within kidney glomeruli. Similarly to E-cadherin, p120 ctn, β-catenin and α-catenin, PLEKHA7 is concentrated in the apical junctional belt, but unlike these adherens junction markers, and similarly to afadin, PLEKHA7 is not localized along the lateral region of polarized epithelial cells. Immunoelectron microscopy definitively establishes that PLEKHA7 is localized at the adherens junctions in colonic epithelial cells, at a mean distance of 28 nm from the plasma membrane. In summary, we show that PLEKHA7 is a cytoplasmic component of the epithelial adherens junction belt, with a subcellular localization and tissue distribution that is distinct from that of ZO-1 and most AJ proteins, and we provide the first description of its distribution and localization in several tissues.

Development of a Qualitative Sequential Immunoassay for Characterizing the Intrinsic Properties of Circulating Cardiac Troponin I

With myocardial infarction (MI), cardiac troponin is released from the heart into circulation, where it can be detected with immunoassays independently quantifying cardiac troponin I (cTnI) or cTnT. There is, however, no single immunoassay that sequentially probes the posttranslational modification status of cTnI or directly characterizes whether circulating cTnI is bound to cTnC and/or cTnT. Here we describe the development of a qualitative immunoassay to directly probe the primary and ternary structure of circulating cTnI through diffractive optics technology (dotLab System, Axela). Anti-cTnI antibody 8I-7 was immobilized on a patterned sensor to capture cTnI. One or more detector antibodies were sequentially introduced to probe for amino acid sequence integrity or phosphorylation status of cTnI, or its association with cTnC and/or cTnT. Respective immunocaptures were recorded as real-time diffractive intensities (DIs), and the DI differences were analyzed. Each immunodetection was independent of the others but was done in a single sequential assay. This diffraction-based immunoassay successfully characterized cTnI. The unamplified assay determined whether cTnI was degraded at N-terminus and/or C-terminus or phosphorylated. Sequential application of multiple detector antibodies without an antibody-stripping step enables real-time interrogation of 5 different epitopes of cTnI, or direct detection of the cTn complex (cTnI-cTnC-cTnT) in a single sequential assay. Finally, this assay was optimized with amplification to directly detect circulating cTnI bound to cTnC and cTnT in serum from an MI patient. The dot Immunoassay is the first qualitative sequential immunoassay to address the direct interactions of the troponin subunits and various modified forms of cTnI.

Effect of troponin I Ser23/24 phosphorylation on Ca2+-sensitivity in human myocardium depends on the phosphorylation background

Protein kinase A (PKA)-mediated phosphorylation of Ser23/24 of cardiac troponin I (cTnI) causes a reduction in Ca2+-sensitivity of force development. This study aimed to determine whether the PKA-induced modulation of the Ca2+-sensitivity is solely due to cTnI phosphorylation or depends on the phosphorylation status of other sarcomeric proteins. Endogenous troponin (cTn) complex in donor cardiomyocytes was partially exchanged (up to 66±1%) with recombinant unphosphorylated human cTn and in failing cells similar exchange was achieved using PKA-(bis)phosphorylated cTn complex. Cardiomyocytes immersed in exchange solution without complex added served as controls. Partial exchange of unphosphorylated cTn complex in donor tissue significantly increased Ca2+-sensitivity (pCa50) to 5.50±0.02 relative to the donor control value (pCa50=5.43±0.04). Exchange in failing tissue with PKA-phosphorylated cTn complex did not change Ca2+-sensitivity relative to the failing control (pCa50=5.60±0.02). Subsequent treatment of the cardiomyocytes with the catalytic subunit of PKA significantly decreased Ca2+-sensitivity in donor and failing tissue. Analysis of phosphorylated cTnI species revealed the same distribution of un-, mono- and bis-phosphorylated cTnI in donor control and in failing tissue exchanged with PKA-phosphorylated cTn complex. Phosphorylation of myosin-binding protein-C in failing tissue was significantly lower compared to donor tissue. These differences in Ca2+-sensitivity in donor and failing cells, despite similar distribution of cTnI species, could be abolished by subsequent PKA-treatment and indicate that other targets of PKA are involved the reduction of Ca2+-sensitivity. Our findings suggest that the sarcomeric phosphorylation background, which is altered in cardiac disease, influences the impact of cTnI Ser23/24 phosphorylation by PKA on Ca2+-sensitivity.

PKA Phosphorylation Has No Effect on the Force-PCA Relationship or Length Dependent Activation Following L48Q cTNC-Tn Exchange in Rat Demembranated Trabeculae

The molecular mechanism behind the Frank-Starling law of the heart is thought to be due in part to changes in myofilament lattice spacing and/or increased myosin proximity to actin binding sites at longer sarcomere lengths (SL). We previously showed that cardiac trabeculae have a left-shifted force-pCa relationship and eliminated SL-dependence of this relationship following passive exchange with cTn containing a mutant (L48Q) cTnC (with enhanced TnC-TnI interaction strength). This result suggests L48Q cTnC reduces the cross-bridge dependent component of thin filament activation. Experiments here were designed to test the hypothesis that cTnI phosphorylation can restore SL dependence in L48Q cTnC exchanged trabeculae by decreasing Tn Ca2+ affinity and/or TnC-TnI interaction strength. Interestingly, PKA treatment did not reduce Ca2+ sensitivity of force or restore SL dependence in L48Q cTnC-cTn exchanged trabeculae. PKA treatment of native and WT cTn exchanged trabeculae right-shifted and also reduced SL dependence of the force-pCa relationship. Other laboratories investigating the effect of PKA on SL dependence have produced discordant results, possibly due to PKA-induced phosphorylation of proteins other than TnI (i.e., MyBP-C and titin) Therefore, planned future experiments will investigate exchange of PKA pre-treated cTn complexes, S23/24D (phosphomimic) cTnI, and S23/24A (non-phosphorylatable) cTnI with WT and L48Q cTnC. Experiments will be coupled with solution measurements cTnI-cTnC affinity using spectrofluorimetry to provide a more detailed understanding of how PKA phosphorylation and cTnI-cTnC interaction strength influence SL-dependence in cardiac muscle. Support provided by NIH HL65497 (MR) and T32 HL07828 (FSK).

Does the DCM Functional Phenotype Predominate over that of HCM and RCM?

In vitro investigations into Hypertrophic Cardiomyopathy (HCM) and Restrictive Cardiomyopathy (RCM) show that mutations in cardiac Troponin T (cTnT) produce a pathogenic state via increase in myofilament Ca2+ sensitivity, whereas mutations in cTnT that cause Dilated Cardiomyopathy (DCM) decrease Ca2+ sensitivity and maximal force. Our aim was to determine whether combinatory mutations of an HCM, RCM and a DCM in cTnT yield an intermediate or dominant functional phenotype that could be correlated with the clinical condition seen in patients. Standard laboratory methods were used for cloning, expression and purification of the WT and mutants: ΔK210 (DCM), I79N (HCM), ΔE96 (RCM), ΔK210/I79N, ΔK210/ΔE96 and ΔE96/I79N. Porcine papillary skinned fibers were displaced with cTnT WT or mutant and reconstituted with HCTnI-TnC. The Ca2+ sensitivity of force development, maximal force and basal force were evaluated. The extent of TnT displacement was analyzed by measuring the unregulated tension at pCa 8.0 after cTnT treatment and none of the mutants showed an inability to displace the native cTn complex. Both double mutants (ΔK210/I79N and ΔK210/ΔE96) containing the DCM mutant showed a rightward shift in the Ca2+ sensitivity with a decrease in maximal force. In addition, the ΔK210 mutation rescued the impaired relaxation produced by the RCM mutation (ΔE96). From the skinned fiber data, ΔK210 has a dominant effect over I79N and DE96 mutations in cTnT. Circular dichroism measurements demonstrated that all three double mutants had lower alpha helical content than WT. In contrast, single mutants I79N (significantly) and DK210 (showed a tendency) to increase alpha helical content. These results suggest that cTnT can exist in multiple conformations that may be responsible for these distinct functional phenotypes. NIH HL-67415 and HL-42325 (JDP), AHA 0825368E (JRP).

A Functional and Structural Study of Troponin C Mutations Related to Hypertrophic Cardiomyopathy

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.

Ala Scanning of the Inhibitory Region of Cardiac Troponin I

In skeletal and cardiac muscles, troponin (Tn), which resides on the thin filament, senses a change in intracellular Ca(2+) concentration. Tn is composed of TnC, TnI, and TnT. Ca(2+) binding to the regulatory domain of TnC removes the inhibitory effect by TnI on the contraction. The inhibitory region of cardiac TnI spans from residue 138 to 149. Upon Ca(2+) activation, the inhibitory region is believed to be released from actin, thus triggering actin-activation of myosin ATPase. In this study, we created a series of Ala-substitution mutants of cTnI to delineate the functional contribution of each amino acid in the inhibitory region to myofilament regulation. We found that most of the point mutations in the inhibitory region reduced the ATPase activity in the presence of Ca(2+), which suggests the same region also acts as an activator of the ATPase. The thin filaments can also be activated by strong myosin head (S1)-actin interactions. The binding of N-ethylmaleimide-treated myosin subfragment 1 (NEM-S1) to actin filaments mimics such strong interactions. Interestingly, in the absence of Ca(2+) NEM-S1-induced activation of S1 ATPase was significantly less with the thin filaments containing TnI(T144A) than that with the wild-type TnI. However, in the presence of Ca(2+), there was little difference in the activation of ATPase activity between these preparations.

Differential effects of phosphorylation of regions of troponin I in modifying cooperative activation of cardiac thin filaments

Ischemia and heart failure are associated with protein kinase C (PKC) dependent phosphorylation of cardiac troponin I (cTnI). We investigated the effect of phosphorylation of cTnI PKC sites S43, S45 and T144 under normal (pH 7.0) and acidic (pH 6.5) conditions on tension in skinned fiber bundles from a mouse heart. To mimic the PKC phosphorylation, we exchanged troponin (cTn) in these fiber bundles with cTn complex containing either cTnI-(S43E/S45E) or cTnI-(T144E). We determined how pseudo-phosphorylation and acidic pH affect activation of thin filaments by strongly bound crossbridges by use of n-ethyl maleimide (NEM-S1) to mimic rigor. We hypothesized that PKC phosphorylation of cTnI amplifies the effect of ischemic/hypoxic conditions to depress myofilament force and Ca2+-responsiveness by reducing the ability of rigor crossbridge to activate force. Pseudo-phosphorylation of cTnI at S43/S45 exacerbated the effect of acidic pH to induce a rightward shift in the Ca2+-tension relation. Under acidic conditions, fibers regulated by cTnI-(S43E/S45E) demonstrated a significant reduction in the ability of NEM-S1 to recruit cycling crossbridges, when compared to controls regulated by cTnI. Similar effects of pseudo-phosphorylation of cTnI-(T144) occurred, but to a lesser extent that those of pseudo-phosphorylation of S43/S45. We conclude that under acidic conditions PKC phosphorylation of cTnI residues at S43/S45 and at T144 is likely to have differential, but significant effects in depressing the ability of both Ca2+ and rigor crossbridges to activate force generation. Although these effects of PKC dependent phosphorylation may be maladaptive in heart failure, they may also spare ATP consumption and be cardio-protective in ischemia.

High Resolution Top-Down MS/MS Reveals Single Amino Acid Sequence Polymorphisms in Rat Cardiac Troponin

Heterotrimeric cardiac troponin (cTn) is a critical component of the thin filament regulatory complex. Two of the three subunits, cTnI and cTnT, are expressed only in cardiac muscle and are widely used in the clinic as serum biomarkers of cardiac injury. cTnI and cTnT are subject to extensive post-translational modification such as proteolysis and phosphorylation, but linking modification patterns to function remains a major challenge. In order to obtain a global view of the state of post-translational modification of cTn, we are performing high resolution top-down mass spectrometry on cTn subunits isolated from native tissues. Whole cTn complexes affinity purified from a single rat heart were analyzed in a 7 Tesla Thermo LTQ-FT-ICR mass spectrometer equipped with an ESI source. High resolution MS spectra of cTn from healthy adult rats showed molecular ions for intact cTnT and cTnI as well as phosphorylation and acetylation patterns similar to human cTnI (Zabrouskov et al., 2008 Mol Cell Proteomics, in press). ‘Shadow peaks’ of similar intensity to parent peaks were detected exhibiting masses of cTnI + 16 Da and cTnT + 128 Da, suggestive of single amino acid polymorphisms. Tandem mass spectrometry (MS/MS) analysis by ECD and CAD fragmentation of intact and protease-digested cTn subunits localized an Ala/Ser polymorphism at residue 7 of cTnI, and an additional Gln within a 3 residue alternative splice site beginning at residue 192 of cTnT. High resolution top-down MS/MS has revealed intriguing heterogeneity not only in the extent of phosphorylation, but also in amino acid sequences of cTnI and cTnT even within a single rat heart. Supported by NIH, AHA, UW-CVRC & Wisconsin Partnership for a Healthy Future.

Effect of Troponin I Ser23/24 Bis-Phosphorylation on Ca2+-Sensitivity is Dependent on PKA Phosphorylation of Other Contractile Proteins

Upon β-adrenergic stimulation, protein kinase A (PKA) enhances cardiac Troponin I (cTnI) phosphorylation at ser23/24. PKA treatment leads to a decrease in myofilament Ca2+-sensitivity. However, the specific effect of PKA-mediated phosphorylation of cTnI in human myocardium is unclear since PKA phosphorylates a broader set of contractile proteins, in particular myosin binding protein C (cMyBP-C).To address this issue, a selective exchange procedure was used in which 50% and 70% of the endogenous cTn complex in permeabilized human cardiomyocytes was exchanged with recombinant unphosphorylated human cTn. Cardiomyocytes isolated from healthy donor hearts showed almost saturated phosphorylation levels at the ser23/24 of cTnI. Endogenous phosphorylated cTn of donor cardiomyocytes (pCa50= 5.45±0.03) was exchanged with 0.5 and 1.0 mg/ml unphosphorylated recombinant human cTn (cTn-U), which resulted in an increase in Ca2+-sensitivity (ΔpCa50=0.08). Subsequent incubation of the cells with PKA reversed Ca2+-sensitivity to baseline levels (pCa50= 5.46±0.03).To study if the effect of PKA-mediated phosphorylation on cTnI ser23/24 depends on phosphorylation of other contractile proteins, failing human cardiac tissue was used in which phosphorylation of cTnI and cMyBP-C is depressed. Cells from failing tissue showed increased Ca2+-sensitivity (pCa50 5.56±0.03) compared to donor cells. Endogenous cTn of failing cardiomyocytes was exchanged with 0.5 and 1.0 mg/ml cTn pre-treated with PKA to fully saturate ser23/24 (cTn-bisP). However, upon exchange with the cTn-bisP complex, Ca2+-sensitivity did not decrease. Subsequent PKA incubation reduced pCa50 back to the level observed in donor myocardium. This indicates that the effect of cTnI ser23/24 bis-phosphorylation on Ca2+-sensitivity is dependent on PKA-mediated phosphorylation of other contractile protein(s). Preliminary protein phosphorylation data point towards the involvement of cMyBP-C.

Kinetic Mechanism of the Ca 2+-Dependent Switch-On and Switch-Off of Cardiac Troponin in Myofibrils

The kinetics of Ca 2+-dependent conformational changes of human cardiac troponin (cTn) were studied on isolated cTn and within the sarcomeric environment of myofibrils. Human cTnC was selectively labeled on cysteine 84 with N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole and reconstituted with cTnI and cTnT to the cTn complex, which was incorporated into guinea pig cardiac myofibrils. These exchanged myofibrils, or the isolated cTn, were rapidly mixed in a stopped-flow apparatus with different [Ca 2+] or the Ca 2+-buffer 1,2-Bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid to determine the kinetics of the switch-on or switch-off, respectively, of cTn. Activation of myofibrils with high [Ca 2+] (pCa 4.6) induced a biphasic fluorescence increase with rate constants of >2000 s −1 and ∼330 s −1, respectively. At low [Ca 2+] (pCa 6.6), the slower rate was reduced to ∼25 s −1, but was still ∼50-fold higher than the rate constant of Ca 2+-induced myofibrillar force development measured in a mechanical setup. Decreasing [Ca 2+] from pCa 5.0–7.9 induced a fluorescence decay with a rate constant of 39 s −1, which was approximately fivefold faster than force relaxation. Modeling the data indicates two sequentially coupled conformational changes of cTnC in myofibrils: 1), rapid Ca 2+-binding ( k B ≈ 120 μM −1 s −1) and dissociation ( k D ≈ 550 s −1); and 2), slower switch-on ( k on = 390s −1) and switch-off ( k off = 36s −1) kinetics. At high [Ca 2+], ∼90% of cTnC is switched on. Both switch-on and switch-off kinetics of incorporated cTn were around fourfold faster than those of isolated cTn. In conclusion, the switch kinetics of cTn are sensitively changed by its structural integration in the sarcomere and directly rate-limit neither cardiac myofibrillar contraction nor relaxation.

CagA-independent disruption of adherence junction complexes involves E-cadherin shedding and implies multiple steps in Helicobacter pylori pathogenicity

Infection with Helicobacter pylori ( H. pylori) leads to depolarization and migration of polarized epithelial cells, both strongly enhanced by injection of the pathogenic factor CagA (cytotoxin-associated gene A) into the host cytoplasm. Depolarization and migration of epithelial cells imply the disruption of cell adhesion junctions (AJs) comprising a protein complex of E-cadherin, β-catenin, p120 ctn, and α-catenin. Here, we analyzed the disintegration of E-cadherin-mediated AJs and demonstrated that loss of E-cadherin-dependent cell–cell contacts is entirely independent of CagA. Upon infection with H. pylori, either wild-type (wt) or a cagA mutant (Δ cagA), interaction between E-cadherin and α-catenin dissociated rapidly, while binding of E-cadherin to β-catenin and p120 ctn was hardly affected. Simultaneously, loss of cell adhesion involved E-cadherin cleavage induced by a bacterial factor secreted by H. pylori. Finally, β-catenin-mediated transcription, a hallmark of many carcinomas, was not activated in H. pylori-infected epithelial cells at this stage of infection. Altogether, our data indicate that H. pylori-induced pathogenesis is a multi-step process initiated by CagA-independent mechanisms. These include proteolytical cleavage of E-cadherin and dissociation of the E-cadherin/β-catenin/p120 ctn complex from the actin cytoskeleton by disrupting binding to α-catenin.

Characterization of the cardiac holotroponin complex reconstituted from native cardiac troponin T and recombinant I and C.

Cardiac troponin I (cTnI), the inhibitory subunit of cardiac troponin (cTn), is phosphorylated by the cAMP-dependent protein kinase A at two adjacently located serine residues within the heart-specific N-terminal elongation. Four different phosphorylation states can be formed. To investigate each monophosphorylated form cTnI mutants, in which each of the two serine residues is replaced by an alanine, were generated. These mutants, as well as the wild-type cardiac troponin I (cTnI-WT) have been expressed in Escherichia coli, purified and characterized by isoelectric focusing, MS and CD-spectroscopy. Monophosphorylation induces conformational changes within cTnI that are different from those induced by bisphosphorylation. Functionality was assessed by measuring the calcium dependence of myosin S1 binding to thin filaments containing reconstituted native, wild-type and mutant cTn complexes. In all cases a functional holotroponin complex was obtained. Upon bisphosphorylation of cTnI-WT the pCa curve was shifted to the right to the same extent as that observed with bisphosphosphorylated native cTnI. However, the absolute values for the midpoints were higher when recombinant cTn subunits were used for reconstitution. Reconstitution itself changed the calcium affinity of cTnC: pCa50-values were higher than those obtained with the native cardiac holotroponin complex. Apparently only bisphosphorylation of cTnI influences the calcium sensitivity of the thin filament, thus monophosphorylation has a function different from that of bisphosphorylation; this function has not yet been identified.

Syntheses, Structures, and Thermal, Transport and Nonlinear Optical Properties of Tyc -Txf Compounds

Abstract The syntheses, electric and structural properties of a series of uncapped C6X4Y4 compounds (tetrakis(n-alkylchalcogeno)- tetrachalcogenafulvalenes; TYC -TXF) and nonlinear electric and optical properties of their CTn complexes are described.