Augmented phosphorylation of cardiac troponin I in hypertensive heart failure.

An altered cardiac myofilament response to activating Ca(2+) is a hallmark of human heart failure. Phosphorylation of cardiac troponin I (cTnI) is critical in modulating contractility and Ca(2+) sensitivity of cardiac muscle. cTnI can be phosphorylated by protein kinase A (PKA) at Ser(22/23) and protein kinase C (PKC) at Ser(22/23), Ser(42/44), and Thr(143). Whereas the functional significance of Ser(22/23) phosphorylation is well understood, the role of other cTnI phosphorylation sites in the regulation of cardiac contractility remains a topic of intense debate, in part, due to the lack of evidence of in vivo phosphorylation. In this study, we utilized top-down high resolution mass spectrometry (MS) combined with immunoaffinity chromatography to determine quantitatively the cTnI phosphorylation changes in spontaneously hypertensive rat (SHR) model of hypertensive heart disease and failure. Our data indicate that cTnI is hyperphosphorylated in the failing SHR myocardium compared with age-matched normotensive Wistar-Kyoto rats. The top-down electron capture dissociation MS unambiguously localized augmented phosphorylation sites to Ser(22/23) and Ser(42/44) in SHR. Enhanced Ser(22/23) phosphorylation was verified by immunoblotting with phospho-specific antibodies. Immunoblot analysis also revealed up-regulation of PKC-α and -δ, decreased PKCε, but no changes in PKA or PKC-ß levels in the SHR myocardium. This provides direct evidence of in vivo phosphorylation of cTnI-Ser(42/44) (PKC-specific) sites in an animal model of hypertensive heart failure, supporting the hypothesis that PKC phosphorylation of cTnI may be maladaptive and potentially associated with cardiac dysfunction.

Cardiac troponin T, a sarcomeric AKAP, tethers protein kinase A at the myofilaments.

Efficient and specific phosphorylation of PKA substrates, elicited in response to ß-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.

Cardiomyopathy-causing deletion K210 in cardiac troponin T alters phosphorylation propensity of sarcomeric proteins.

Ca(2+) desensitization of myofilaments is indicated as a primary mechanism for the pathogenesis of familial dilated cardiomyopathy (DCM) associated with the deletion of lysine 210 (DeltaK210) in cardiac troponin T (cTnT). DeltaK210 knock-in mice closely recapitulate the clinical phenotypes documented in patients with this mutation. Considerable evidence supports the proposition that phosphorylation of cardiac sarcomeric proteins is a key modulator of function and may exacerbate the effect of the deletion. In this study we investigate the impact of K210 deletion on phosphorylation propensity of sarcomeric proteins. Analysis of cardiac myofibrils isolated from DeltaK210 hearts identified a decrease in phosphorylation of cTnI (46%), cTnT (30%) and MyBP-C (32%) compared with wild-type controls. Interestingly, immunoblot analyses with phospho-specific antibodies show augmented phosphorylation of cTnT-Thr(203) (28%) and decreased phosphorylation of cTnI-Ser(23/24) (41%) in mutant myocardium. In vitro kinase assays indicate that DeltaK210 increases phosphorylation propensity of cTnT-Thr(203) three-fold, without changing cTnI-Ser(23/24) phosphorylation. Molecular modeling of cTnT-DeltaK210 structure reveals changes in the electrostatic environment of cTnT helix (residues 203-224) that lead to a more basic environment around Thr(203), which may explain the enhanced PKC-dependent phosphorylation. In addition, yeast two-hybrid assays indicate that cTnT-DeltaK210 binds stronger to cTnI compared with cTnT-wt. Collectively, our observations suggest that cardiomyopathy-causing DeltaK210 has far-reaching effects influencing cTnI-cTnT binding and posttranslational modifications of key sarcomeric proteins.

Impact of cardiac troponin T N-terminal deletion and phosphorylation on myofilament function.

Cardiac troponin T (cTnT) is a phosphoprotein that modulates cardiac muscle contraction through its extensive and diverse interactions with neighboring thin filament proteins. Its N-terminal half is the "glue" that anchors the troponin complex to tropomyosin-actin. Until now, studies aimed at investigating the role of the N-terminal tail region have not considered the effects of phosphorylation. To understand better the regulatory role of the N-terminal tail region of phosphorylated cTnT, we investigated the functional effects of N-terminal deletion (amino acids 1-91) and phosphorylation on Ca(2+) dependence of myofilament isometric force production, isometric ATPase rate, and thin filament sliding speed. Chemomechanical profiles were assessed in detergent permeabilized fiber preparations where the native troponin (cTn) was exchanged with recombinant cTn engineered to contain modified cTnT (truncated, phosphorylated) in the presence of wild-type cTnI and cTnC. Removal of the cTnT N-terminal amino acids 1-91 (cTnT-del) enhances myofilament responsiveness to nonsaturating Ca(2+) levels (the physiological range in cardiac myocytes). However, at saturating Ca(2+) levels, there is a reduction in isometric tension and ATPase rate. On one hand, phosphorylation of cTnT-del attenuates the sensitizing effect induced by truncation of the N-terminal tail, "resetting" myofilament Ca(2+) responsiveness back to control levels. On the other hand, it impairs isometric tension development and ATPase rate. Interestingly, phosphorylation of cTnT (cTnT-P) differentially regulates tension cost (an index of cross-bridge cycling rate): increased by cTn-del-P and decreased by intact cTn-wt-P. Like the isometric fiber data, sliding speed of thin filaments regulated by cTn-del is more sensitive to Ca(2+) compared with cTn-wt. Phosphorylation of cTnT (whether cTnT-del or -wt) depresses sliding speed and is associated with Ca(2+) desensitization of thin filament sliding speed.

Funding opportunities for investigators in the early stages of career development.

Many sources of advice and guidance are available to the early career investigator. Generally, mentors serve as the primary source of information, although program and review officers are the most underutilized resources. This article organizes these opportunities to enable early career investigators to plot a rational trajectory for career success. A list of the major agencies that provide grant support for early career investigators is included. In addition, funding opportunities are organized on the basis of the stage in career development pathway and the type of terminal degree.