Phosphorylation Of Cardiac Troponin Research Articles

The Interplay between S-Glutathionylation and Phosphorylation of Cardiac Troponin I and Myosin Binding Protein C in End-Stage Human Failing Hearts.

Oxidative stress is defined as an imbalance between the antioxidant defense system and the production of reactive oxygen species (ROS). At low levels, ROS are involved in the regulation of redox signaling for cell protection. However, upon chronical increase in oxidative stress, cell damage occurs, due to protein, DNA and lipid oxidation. Here, we investigated the oxidative modifications of myofilament proteins, and their role in modulating cardiomyocyte function in end-stage human failing hearts. We found altered maximum Ca2+-activated tension and Ca2+ sensitivity of force production of skinned single cardiomyocytes in end-stage human failing hearts compared to non-failing hearts, which was corrected upon treatment with reduced glutathione enzyme. This was accompanied by the increased oxidation of troponin I and myosin binding protein C, and decreased levels of protein kinases A (PKA)- and C (PKC)-mediated phosphorylation of both proteins. The Ca2+ sensitivity and maximal tension correlated strongly with the myofilament oxidation levels, hypo-phosphorylation, and oxidative stress parameters that were measured in all the samples. Furthermore, we detected elevated titin-based myocardial stiffness in HF myocytes, which was reversed by PKA and reduced glutathione enzyme treatment. Finally, many oxidative stress and inflammation parameters were significantly elevated in failing hearts compared to non-failing hearts, and corrected upon treatment with the anti-oxidant GSH enzyme. Here, we provide evidence that the altered mechanical properties of failing human cardiomyocytes are partially due to phosphorylation, S-glutathionylation, and the interplay between the two post-translational modifications, which contribute to the development of heart failure.

Abstract 405: Functional Maturation of Human iPSC-Derived Cardiomyocytes by Prolonged 3D Culture in Engineered Cardiac Tissue Constructs

The advent of human induced pluripotent stem cell (hiPSC) technology has revolutionized the way we study inherited diseases, as it allows us to study the effect of mutations in a human context. This is particularly true in the heart were species-specific differences has dramatic effects on heart rate, and expression of protein isoforms involved in excitation-contraction coupling. A major drawback of using hiPSCs is, however that cardiomyocytes derived from hiPSC (hiPSC-CM) are relatively immature, limiting their utility as a tool to study adult onset cardiac diseases. We postulated that culturing hiPSC-CM in a 3D environment in engineered cardiac tissue constructs (ECT) would promote hiPSC-CM maturation. ECTs were generated from lactate-selected day 30 hiPSCs that were mixed with isogenic hiPSC derived cardiac fibroblasts in a 10:1 ratio. Cells were mixed with fibrinogen and thrombin and seeded into molds under vacuum in FlexCell dishes to form ECT. ECTs were harvested 14-21 or 42-52 days later and subjected to functional testing, transcriptional analysis and top-down mass-spectrometry of sarcomeric proteins to assess maturation. Functionally, we found that prolonged culture of hiPSCs in ECT resulted in increased calcium transient amplitude (0.856 vs. 0.462; P < 0.01) and an acceleration of calcium kinetics (calcium release time 67.7 ms vs. 109.3 ms; P < 0.01 and calcium decay time 148.3 ms vs. 188.0 ms; P < 0.05). This acceleration of calcium kinetics with time in culture was also true for twitch force kinetics (time to peak twitch force 178.6 ms vs. 208.0 ms; P < 0.05). Furthermore, beta adrenergic stimulation had a much greater effect on twitch kinetics in older ECTs (reduction in contraction time of 26.5% vs. 7.1%; P < 0.01). Transcriptional analysis revealed an increase in expression level of the beta 1 adrenergic receptor in older ECTs, while top-down mass spectrometry showed increased expression and phosphorylation of cardiac troponin I (cTnI) and mono-phosphorylated cTnI, as well as decreased phosphorylation of alpha-tropomyocin, all markers of myocardial maturation. Taken together, these data supports our hypothesis that prolonged culture of hiPSC-CM in ECT promotes maturation of the calcium handling system and the contractile apparatus.

Mechanistic role of the CREB-regulated transcription coactivator 1 in cardiac hypertrophy

The sympathetic nervous system is the main stimulator of cardiac function. While acute activation of the β-adrenoceptors exerts positive inotropic and lusitropic effects by increasing cAMP and Ca2+, chronically enhanced sympathetic tone with changed β-adrenergic signaling leads to alterations of gene expression and remodeling. The CREB-regulated transcription coactivator 1 (CRTC1) is activated by cAMP and Ca2+. In the present study, the regulation of CRTC1 in cardiomyocytes and its effect on cardiac function and growth was investigated. In cardiomyocytes, isoprenaline induced dephosphorylation, and thus activation of CRTC1, which was prevented by propranolol. Crtc1-deficient mice exhibited left ventricular dysfunction, hypertrophy and enlarged cardiomyocytes. However, isoprenaline-induced contractility of isolated trabeculae or phosphorylation of cardiac troponin I, cardiac myosin-binding protein C, phospholamban, and ryanodine receptor were not altered, suggesting that cardiac dysfunction was due to the global lack of Crtc1. The mRNA and protein levels of the Gαq GTPase activating protein regulator of G-protein signaling 2 (RGS2) were lower in hearts of Crtc1-deficient mice. Chromatin immunoprecipitation and reporter gene assays showed stimulation of the Rgs2 promoter by CRTC1. In Crtc1-deficient cardiomyocytes, phosphorylation of the Gαq-downstream kinase ERK was enhanced. CRTC1 content was higher in cardiac tissue from patients with aortic stenosis or hypertrophic cardiomyopathy and from two murine models mimicking these diseases. These data suggest that increased CRTC1 in maladaptive hypertrophy presents a compensatory mechanism to delay disease progression in part by enhancing Rgs2 gene transcription. Furthermore, the present study demonstrates an important role of CRTC1 in the regulation of cardiac function and growth.

Pim-1 Kinase Phosphorylates Cardiac Troponin I and Regulates Cardiac Myofilament Function

Background/Aims: Pim-1 is a serine/threonine kinase that is highly expressed in the heart, and exerts potent cardiac protective effects through enhancing survival, proliferation, and regeneration of cardiomyocytes. Its myocardial specific substrates, however, remain unknown. In the present study, we aim to investigate whether Pim-1 modulates myofilament activity through phosphorylation of cardiac troponin I (cTnI), a key component in regulating myofilament function in the heart. Methods: Coimmunoprecipitation and immunofluorescent assays were employed to investigate the interaction of Pim-1 with cTnI in cardiomyocytes. Biochemical, site directed mutagenesis, and mass spectrometric analyses were utilized to identify the phosphorylation sites of Pim1 in cTnI. Myofilament functional assay using skinned cardiac fiber was used to assess the effect of Pim1-mediated phosphorylation on cardiac myofilament activity. Lastly, the functional significance of Pim1-mediated cTnI in heart disease was determined in diabetic mice. Results: We found that Pim-1 specifically interacts with cTnI in cardiomyocytes and this interaction leads to Pim1-mediated cTnI phosphorylation, predominantly at Ser23/24 and Ser150. Furthermore, our functional assay demonstrated that Pim-1 induces a robust phosphorylation of cTnI within the troponin complex, thus leading to a decreased Ca2+ sensitivity. Insulin-like growth factor 1 (IGF-1), a peptide growth factor that has been shown to stimulate myocardial contractility, markedly induces cTnI phosphorylation at Ser23/24 and Ser150 through increasing Pim-1 expression in cardiomyocytes. In a high-fat diabetic mice model, the expression of Pim1 in the heart is significantly decreased, which is accompanied by a decreased phosphorylation of cTnI at Ser23/24 and Ser150, further implicating the pathological significance of the Pim1/cTnI axis in the development of diabetic cardiomyopathy. Conclusion: Our results demonstrate that Pim-1 is a novel kinase that phosphorylates cTnI primarily at Ser23/24 and Ser150 in cardiomyocytes, which in turn may modulate myofilament function under a variety of physiological and pathophysiological conditions.

Abstract 15554: Heart Failure-Related Hyperphosphorylation of Ser199 on Cardiac Troponin I Causes Divergent Effects on Cardiac Function at Baseline and During Ischemia-Reperfusion

Background: Phosphorylation of cardiac troponin I (cTnI) on Ser199 is significantly increased in human heart failure (HF), however its cardiac impact in vivo remains unknown. In addition, selective proteolysis of cTnI is a marker of myocardial injury and contributes to cardiac dysfunction in ischemia/reperfusion (I/R), and Ser199 phosphorylation affects cTnI proteolysis in vitro, thus whether Ser199 phosphorylation affects cardiac performance during I/R is of great interest. Methods and Results: We generated transgenic mice (TgS200D) carrying cTnI S200D mutation to mimic the site-specific hyperphosphorylation of murine Ser200 (equivalent to human Ser199). Cardiac function was first assessed in vivo using echocardiography. TgS200D mice demonstrated unaltered ejection fraction (EF), significantly prolonged isovolumic relaxation time, and normal left ventricular (LV) chamber size and wall thickness (n=10). LV pressure-volume studies showed preserved dp/dtmax, significantly decreased -dp/dtmin and increased relaxation time constant in TgS200D when compared to non-transgenic (NTg) control group at baseline (n=10). With increasing heart rates, the two groups showed similar enhancement in both systolic and diastolic function, suggesting the positive force-frequency relation is preserved in TgS200D (n=5). After isoproterenol injection (i.v. 40ng/min/kg), although a comparable increase in dp/dtmax was observed in both groups, there was no enhancement of -dp/dtmin in TgS200D in contrast to 15% of increase in NTg (n=5, p=0.019). After ischemia (30 min)/reperfusion (60 min), LV developed pressure was recovered by ~90% in isolated TgS200D hearts but only ~40% in NTg (n=5, p=0.003). Immunoblotting detected cTnI cleavage only in NTg hearts. Conclusions: Our results indicate that hyperphosphorylation of cTnI Ser199 1) impairs basal diastolic function but preserves systolic function, without LV hypertrophy or dilation; 2) blunts cardiac lusitropic response to β-adrenergic stimulation with isoproterenol; 3) protects heart against I/R injury, likely via preventing cTnI degradation. We propose a dual role of cTnI Ser199 hyperphosphorylaiton: contributing to HF with preserved EF but protecting heart during I/R.

Comprehensive assessment of chamber-specific and transmural heterogeneity in myofilament protein phosphorylation by top-down mass spectrometry

The heart is characterized by a remarkable degree of heterogeneity, the basis of which is a subject of active investigation. Myofilament protein post-translational modifications (PTMs) represent a critical mechanism regulating cardiac contractility, and emerging evidence shows that pathological cardiac conditions induce contractile heterogeneity that correlates with transmural variations in the modification status of myofilament proteins. Nevertheless, whether there exists basal heterogeneity in myofilament protein PTMs in the heart remains unclear. Here we have systematically assessed chamber-specific and transmural variations in myofilament protein PTMs, specifically, the phosphorylation of cardiac troponin I (cTnI), cardiac troponin T (cTnT), tropomyosin (Tpm), and myosin light chain 2 (MLC2). We show that the phosphorylation of cTnI and αTm vary in the different chambers of the heart, whereas the phosphorylation of MLC2 and cTnT does not. In contrast, no significant transmural differences were observed in the phosphorylation of any of the myofilament proteins analyzed. These results highlight the importance of appropriate tissue sampling—particularly for studies aimed at elucidating disease mechanisms and biomarker discovery—in order to minimize potential variations arising from basal heterogeneity in myofilament PTMs in the heart.

A novel phosphorylation site, Serine 199, in the C-terminus of cardiac troponin I regulates calcium sensitivity and susceptibility to calpain-induced proteolysis

Phosphorylation of cardiac troponin I (cTnI) by protein kinase C (PKC) is implicated in cardiac dysfunction. Recently, Serine 199 (Ser199) was identified as a target for PKC phosphorylation and increased Ser199 phosphorylation occurs in end-stage failing compared with non-failing human myocardium. The functional consequences of cTnI-Ser199 phosphorylation in the heart are unknown. Therefore, we investigated the impact of phosphorylation of cTnI-Ser199 on myofilament function in human cardiac tissue and the susceptibility of cTnI to proteolysis. cTnI-Ser199 was replaced by aspartic acid (199D) or alanine (199A) to mimic phosphorylation and dephosphorylation, respectively, with recombinant wild-type (Wt) cTn as a negative control. Force development was measured at various [Ca2+] and at sarcomere lengths of 1.8 and 2.2μm in demembranated cardiomyocytes in which endogenous cTn complex was exchanged with the recombinant human cTn complexes. In idiopathic dilated cardiomyopathy samples, myofilament Ca2+-sensitivity (pCa50) at 2.2μm was significantly higher in 199D (pCa50=5.79±0.01) compared to 199A (pCa50=5.65±0.01) and Wt (pCa50=5.66±0.02) at ~63% cTn exchange. Myofilament Ca2+-sensitivity was significantly higher even with only 5.9±2.5% 199D exchange compared to 199A, and saturated at 12.3±2.6% 199D exchange. Ser199 pseudo-phosphorylation decreased cTnI binding to both actin and actin-tropomyosin. Moreover, altered susceptibility of cTnI to proteolysis by calpain I was found when Ser199 was pseudo-phosphorylated. Our data demonstrate that low levels of cTnI-Ser199 pseudo-phosphorylation (~6%) increase myofilament Ca2+-sensitivity in human cardiomyocytes, most likely by decreasing the binding affinity of cTnI for actin-tropomyosin. In addition, cTnI-Ser199 pseudo-phosphorylation or mutation regulates calpain I mediated proteolysis of cTnI.

Pathogenesis associated with a restrictive cardiomyopathy mutant in cardiac troponin T is due to reduced protein stability and greatly increased myofilament Ca2+ sensitivity

BackgroundDilated and hypertrophic cardiomyopathy mutations in troponin can blunt effects of protein kinase A (PKA) phosphorylation of cardiac troponin I (cTnI), decreasing myofilament Ca2+-sensitivity; however this effect has never been tested for restrictive cardiomyopathy (RCM) mutants. This study explores whether an RCM cardiac troponin T mutant (cTnT-ΔE96) interferes with convergent PKA regulation and if TnT instability contributes to greatly enhanced Ca2+-sensitivity in skinned fibers. MethodsForce of contraction in skinned cardiac porcine fiber and spectroscopic studies were performed. ResultsA decrease of −0.26 and −0.25 pCa units in Ca2+-sensitivity of contraction after PKA incubation was observed for skinned fibers incorporated with WT or cTnT-ΔE96, respectively. To further assess whether cTnT-ΔE96 interferes solely with transmission of cTnI phosphorylation effects, skinned fibers were reconstituted with PKA pseudo-phosphorylated cTnI (cTnI-SS/DD.cTnC). Fibers displaced with cTnT-WT, reconstituted with cTnI-SS/DD.cTnC decreased Ca2+-sensitivity of force (pCa50=5.61) compared to control cTnI-WT.cTnC (pCa50=5.75), similarly affecting cTnT-ΔE96 (pCa50=6.03) compared to control \\cTnI-WT.cTnC (pCa50=6.14). Fluorescence studies measuring cTnCIAANS Ca2+-affinity changes due to cTnT-ΔE96 indicated that higher complexity (thin filament) better recapitulates skinned fiber Ca2+ sensitive changes. Circular dichroism revealed reduced α-helicity and earlier thermal unfolding for cTnT-ΔE96 compared to WT. ConclusionsAlthough ineffective in decreasing myofilament Ca2+-sensitivity to normal levels, cTnT-ΔE96 does not interfere with PKA cTnI phosphorylation mediated effects; 2) cTnT-ΔE96 requires actin to increase cTnC Ca2+-affinity; and 3) deletion of E96 reduces cTnT stability, likely disrupting crucial thin filament interactions. General significanceThe pathological effect of cTnT-ΔE96 is largely manifested by dramatic myofilament Ca2+-sensitization which still persists even after PKA phosphorylation mediated Ca2+-desensitization.

Mechanisms that regulate PKCδ‐dependent phosphorylation of cardiac troponin I: the role of the C2 domain and ATP‐binding loop phosphorylation S357 (1081.2)

PKCδ's diverse roles in cell growth, survival, and injury have been attributed to stimulus‐specific differences in PKCδ‐dependent signaling responses. PKCδ exerts membrane‐delimited actions following activation by agonists that stimulate phosphoinositide hydrolysis. PKCδ is released from membranes as a Y311‐phosphorylated enzyme that displays a high level of lipid‐independent activity and altered substrate specificity during oxidative stress. We previously showed that PKCδ phosphorylates cardiac troponin I (cTnI) at S23/S24 when it is allosterically‐activated by lipid‐cofactors and that PKCδ acquires activity toward an additional site on cTnI (T144) during oxidative stress when it is Y311‐phosphorylated by Src. This study identifies an interaction between PKCδ’s phosphotyrosine‐binding C2 domain and the Y311‐phosphorylated hinge region (which resides in a C2 domain consensus‐binding motif) that controls PKCδ's enzymology indirectly by regulating ATP‐positioning loop phosphorylation at S357. Mutagenesis studies show that PKCδ‐S357A is a lipid‐independent enzyme that displays high levels of cTnI‐S23/S24 and T141 kinase activity. In contrast, phosphomimetic or bulky substitutions at S357 prevent cTnI phosphorylation at T141 (but not S23/S24) due to an effect on the enzyme’s phosphoacceptor site selectivity. S‐A substitutions at cognate sites in the highly‐conserved ATP‐binding loops of PKCα and PKA increase catalytic activity, but do not alter phosphoacceptor site selectivity. The C2 domain‐pY311 docking interaction that controls ATP‐binding loop phosphorylation represents a novel structural determinant of PKCδ catalytic activity.Grant Funding Source: Supported by HL77860

Nuclear Localization of α1A‐Adrenergic Receptors Is Required for Signaling in Cardiac Myocytes: An “Inside‐Out” α1‐AR Signaling Pathway

BackgroundRecent studies indicate that α1‐adrenergic receptors (α1‐ARs) are cardioprotective by preventing cardiac myocyte death and augmenting contractility in heart failure. Although G‐protein‐coupled receptors are assumed to localize to and signal at the plasma membrane, we previously demonstrated that endogenous α1‐ARs localize to the nuclei in adult cardiac myocytes. However, the functional consequence of this nuclear localization remains unclear. Here, we attempted to reconcile nuclear localization of α1‐ARs with their physiologic function by examining α1‐AR‐induced contractility in adult cardiac myocytes.Methods and ResultsBy measuring shortening in unloaded, cultured adult cardiac myocytes, we found that the α1A‐subtype regulated contractility through phosphorylation of cardiac troponin I (cTnI) at the protein kinase C (PKC) site, threonine 144. Reconstitution of an α1A‐subtype nuclear localization mutant in cardiac myocytes lacking α1‐ARs failed to rescue nuclear α1A‐mediated phosphorylation of cTnI and myocyte contractility. Leptomycin B, the nuclear export inhibitor, also blocked α1A‐mediated phosphorylation of cTnI. These data indicate that α1‐AR signaling originates in the nucleus. Consistent with these observations, we localized the α1A‐subtype to the inner nuclear membrane, identified PKCα, δ, and ε in the nucleus, and found that α1‐ARs activate PKCδ in nuclei isolated from adult cardiac myocytes. Finally, we found that a PKCδ nuclear localization mutant blunted α1‐induced phosphorylation of cTnI.ConclusionsTogether, our data identify a novel, “inside‐out” nuclear α1A‐subtype/PKCδ/cTnI‐signaling pathway that regulates contractile function in adult cardiac myocytes. Importantly, these data help resolve the discrepancy between nuclear localization of α1‐ARs and α1‐AR‐mediated physiologic function.

Characterization and validation of new tools for measuring site-specific cardiac troponin I phosphorylation

Phosphorylation of cardiac troponin I is a well established mechanism by which cardiac contractility is modulated. However, there are a number of phosphorylation sites on TnI which contribute singly or in combination to influence cardiac function. Accordingly, methods for accurately measuring site-specific TnI phosphorylation are needed. Currently, two strategies are employed: mass spectrometry, which is costly, difficult and has a low throughput; and Western blotting using phospho-specific antibodies, which is limited by the availability of reagents. In this report, we describe a cohort of new site-specific TnI phosphoantibodies, generated against physiologically relevant phosphorylation sites, that are superior to the current commercially available antibodies: to phospho-serine 22/23 which shows a >5-fold phospho-specificity for phosphorylated TnI; to phospho-serine 43, which has >3-fold phospho-specificity for phosphorylated TnI; and phospho-serine150 which has >2-fold phospho-specificity for phosphorylated TnI. These new antibodies demonstrated greater sensitivity and specificity for the phosphorylated TnI than the most widely used commercially available reagents. For example, at a protein load of 20μg of total cardiac extract, a commercially available antibody recognized both phosphorylated and dephosphorylated TnI to the same degree. At the same protein load our phospho-serine 22/23 antibody exhibited no cross-reactivity with dephosphorylated TnI. These new tools should allow a more accurate assessment and a better understanding of the role of TnI phosphorylation in the response of the heart to pathologic stress.

The β-arrestin-biased ligand TRV120023 inhibits angiotensin II-induced cardiac hypertrophy while preserving enhanced myofilament response to calcium

In the present study, we compared the cardioprotective effects of TRV120023, a novel angiotensin II (ANG II) type 1 receptor (AT1R) ligand, which blocks G protein coupling but stimulates β-arrestin signaling, against treatment with losartan, a conventional AT1R blocker in the treatment of cardiac hypertrophy and regulation of myofilament activity and phosphorylation. Rats were subjected to 3 wk of treatment with saline, ANG II, ANG II + losartan, ANG II + TRV120023, or TRV120023 alone. ANG II induced increased left ventricular mass compared with rats that received ANG II + losartan or ANG II + TRV120023. Compared with saline controls, ANG II induced a significant increase in pCa50 and maximum Ca(2+)-activated myofilament tension but reduced the Hill coefficient (nH). TRV120023 increased maximum tension and pCa50, although to lesser extent than ANG II. In contrast to ANG II, TRV120023 increased nH. Losartan blocked the effects of ANG II on pCa50 and nH and reduced maximum tension below that of saline controls. ANG II + TRV120023 showed responses similar to those of TRV120023 alone; compared with ANG II + losartan, ANG II + TRV120023 preserved maximum tension and increased both pCa50 and cooperativity. Tropomyosin phosphorylation was lower in myofilaments from saline-treated hearts compared with the other groups. Phosphorylation of cardiac troponin I was significantly reduced in ANG II + TRV120023 and TRV120023 groups versus saline controls, and myosin-binding protein C phosphorylation at Ser(282) was unaffected by ANG II or losartan but significantly reduced with TRV120023 treatment compared with all other groups. Our data indicate that TRV120023-related promotion of β-arrestin signaling and enhanced contractility involves a mechanism promoting the myofilament response to Ca(2+) via altered protein phosphorylation. Selective activation of β-arrestin-dependent pathways may provide advantages over conventional AT1R blockers.

TNNI3K is a novel mediator of myofilament function and phosphorylates cardiac troponin I

The phosphorylation of cardiac troponin I (cTnI) plays an important role in the contractile dysfunction associated with heart failure. Human cardiac troponin I-interacting kinase (TNNI3K) is a novel cardiac-specific functional kinase that can bind to cTnI in a yeast two-hybrid screen. The purpose of this study was to investigate whether TNNI3K can phosphorylate cTnI at specific sites and to examine whether the phosphorylation of cTnI caused by TNNI3K can regulate cardiac myofilament contractile function. Co-immunoprecipitation was performed to confirm that TNNI3K could interact with cTnI. Kinase assays further indicated that TNNI3K did not phosphorylate cTnI at Ser23/24 and Ser44, but directly phosphorylated Ser43 and Thr143 in vitro. The results obtained for adult rat cardiomyocytes also indicated that enhanced phosphorylation of cTnI at Ser43 and Thr143 correlated with rTNNI3K (rat TNNI3K) overexpression, and phosphorylation was reduced when rTNNI3K was knocked down. To determine the contractile function modulated by TNNI3K-mediated phosphorylation of cTnI, cardiomyocyte contraction was studied in adult rat ventricular myocytes. The contraction of cardiomyocytes increased with rTNNI3K overexpression and decreased with rTNNI3K knockdown. We conclude that TNNI3K may be a novel mediator of cTnI phosphorylation and contribute to the regulation of cardiac myofilament contraction function.

Myofilament incorporation and contractile function after gene transfer of cardiac troponin I Ser43/45Ala

Phosphorylation of cardiac troponin I serines 43/45 (cTnISer43/45) by protein kinase C (PKC) is associated with cardiac dysfunction and yet there is disagreement about the role this cluster plays in modulating contractile performance. The present study evaluates the impact of phospho-null Ala substitutions at Ser43/45 (cTnISer43/45Ala) on contractile performance in intact myocytes. Viral-based gene transfer of cardiac troponin I (cTnI) or cTnISer43/45Ala resulted in time-dependent increases in expression, with 70–80% of endogenous cTnI replaced within 4days. Western analysis of intact and permeabilized myocytes along with immunohistochemistry showed each exogenous cTnI was incorporated into the sarcomere of myocytes. In contractile function studies, there were no differences in shortening and re-lengthening for cTnI and cTnISer43/45Ala-expressing myocytes 2days after gene transfer. However, more extensive replacement with cTnISer43/45Ala after 4days diminished peak shortening amplitude and accelerated re-lengthening measured as the time to 50% re-lengthening (TTR50%). A decrease in myofilament Ca2+ sensitivity of tension also was observed in permeabilized myocytes expressing cTnISer43/45Ala and is consistent with accelerated re-lengthening observed in intact myocytes under basal conditions. Phosphorylation of cTnI Ser23/24 and the Ca2+ transient were not changed in these myocytes. These results demonstrate extensive sarcomere expression of cTnISer43/45Ala directly modulates myofilament function under basal conditions. In further work, the accelerated re-lengthening observed in control or cTnI-expressing myocytes treated with the PKC agonist, endothelin-1 (ET, 10nM) was slowed in myocytes expressing cTnISer43/45Ala. This outcome may indicate Ser43/45 is targeted for phosphorylation by ET-activated PKC and/or influences transduction of this agonist-activated response.

Structural and Kinetic Studies using FRET: Impact of Pseudo-Pkc Phosphorylation of Cardiac Troponin T on Calcium-Activated Thin Filament Regulation

Regulation of myocardial contractility is influenced by numerous signaling agents. One of the key methods for modification of cardiomyocyte function is phosphorylation of cardiac troponin (cTn), the Ca2+-dependent regulatory switch that controls thin filament activation, by isoforms of protein kinase C (PKC). PKC has been shown to phosphorylate the tropomyosin binding subunit of cTn (cTnT) at Thr-195, Thr-199, Thr-204 and Thr-285 (rat cTnT). The effect of these phosphorylations on cTn structure and dynamics remains largely unknown. To ascertain the site specific effects of PKC phosphorylation of cTnT on thin filament regulation, pseudo-phosphorylation of cTnT was combined with Forster resonance energy transfer (FRET) to monitor the transition between the Ca2+ and Mg2+ states of cTn. Single-cysteine cTnI mutants were labeled with IAEDENS at either Cys-151 or Cys-167, and a single-cysteine cTnC mutant was labeled at Cys-89 with DDPM. Different combinations of mutant cTnT and fluorophore labeled cTnI and cTnC were then incorporated into reconstituted thin filaments, which were subjected to calcium titrations, time resolved FRET structural measurements, and stopped flow kinetic measurements. Titrations showed a consistent pseudo-phosphorylation dependent increase in pCa50 values, and kinetics experiments showed decreased rates of Ca2+ dissociation in pseudo-phosphorylated mutants. Additionally, Mg2+ distances between Cys-167 of cTnI and Cys-89 of cTnC were reduced in the presence of pseudo-phosphorylation. Thus our results suggested that pseudo-phosphorylation of cTnT leads to sensitization of the Ca2+-dependent conformational transitions involved in cTn regulatory switching. However, a prior in situ study demonstrated a pseudo-phosphorylation dependent decrease in EC50 (JBC vol. 278, pp. 35135-44). These results may suggest PKC mediated phosphorylation of cTnT may represent a post-translational modification where cTn regulatory switching is sensitized to Ca2+, but also partially uncoupled from Tm movement.

Abstract 312: Troponin I Phosphorylation Crosstalk

The troponin (Tn) complex is a critical regulatory and integrative hub for myofilament post-translational modifications that regulate cardiac contraction. Beta-adrenergic-induced protein kinase A (PKA) phosphorylation of cardiac troponin I (cTnI) at Ser-23/24 is a major myofilament cardiac contractile modulator. In addition to this PKA-induced phosphorylation, cTnI can be simultaneously phosphorylated at a number of other residues. To fully understand the molecular mechanisms that underlie cardiac muscle regulation at the myofilament level, cTnI phosphorylations must be studied as integrated events. As an initial step towards understanding the integration of these events, we employed a phosphomimetic approach to investigate the effect of cTnI Ser-150 pseudo-phosphorylation (TnI-S150D) on cTnI PKA pseudo-phosphorylation (TnI-S23/24D) function within the thin filament. The combinatorial effect of these phosphorylation events were determined by measuring the binding of calcium to troponin C in thin filaments reconstituted with human Tn. The steady-state calcium sensitivity of TnC was increased ∼2.3 fold in thin filaments reconstituted with TnI-S150D containing Tn, while TnI-S23/24D containing filaments exhibited an ∼3.9 fold decrease of calcium sensitivity. Importantly, in the presence of both cTnI pseudo-phosphorylations (TnI-S23/24/150D) calcium binding was not different from that of wild-type. Likewise, calcium dissociation from thin filaments reconstituted with TnI-S23/24D was increased by ∼3.5 fold, while TnI-S150D containing filaments demonstrated a decrease in calcium dissociation of ∼2.3 fold compared to wild-type filaments. Interestingly the combined pseudo-phosphorylations of TnI-S23/24/150D only blunted TnI-23/24D calcium dissociation kinetics to ∼1.2 fold of the TnI-S23/24D filaments alone. These findings demonstrate the integrated effects of cTnI site-specific phosphorylation crosstalk to affect the function of cTnI PKA-induced phosphorylation. These data further highlight the importance of understanding the integrated role of Tn phosphorylation crosstalk on cardiac contractile regulation.

Effect of Hypertrophic Cardiomyopathy-Linked Troponin C Mutations on the Response of Reconstituted Thin Filaments to Calcium upon Troponin I Phosphorylation

The objective of this work was to investigate the effect of hypertrophic cardiomyopathy-linked A8V and E134D mutations in cardiac troponin C (cTnC) on the response of reconstituted thin filaments to calcium upon phosphorylation of cardiac troponin I (cTnI) by protein kinase A. The phosphorylation of cTnI at protein kinase A sites was mimicked by the S22D/S23D double mutation in cTnI. Our results demonstrate that the A8V and E134D mutations had no effect on the extent of calcium desensitization of reconstituted thin filaments induced by cTnI pseudophosphorylation. However, the A8V mutation enhanced the effect of cTnI pseudophosphorylation on the rate of dissociation of calcium from reconstituted thin filaments and on the calcium dependence of actomyosin ATPase. Consequently, while the A8V mutation still led to a slower rate of dissociation of calcium from reconstituted thin filaments upon pseudophosphorylation of cTnI, the ability of the A8V mutation to decrease the rate of calcium dissociation was weakened. In addition, the ability of the A8V mutation to sensitize actomyosin ATPase to calcium was weakened after cTnI was replaced by the phosphorylation mimetic of cTnI. Consistent with the hypothesis that the E134D mutation is benign, it exerted a minor to no effect on the rate of dissociation of calcium from reconstituted thin filaments or on the calcium sensitivity of actomyosin ATPase, regardless of the cTnI phosphorylation status. In conclusion, our study enhances our understanding of how cardiomyopathy-linked cTnC mutations affect the response of reconstituted thin filaments to calcium upon cTnI phosphorylation.

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-β 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.

Troponin Phosphorylation Crosstalk

The troponin (Tn) complex is a critical regulatory and central integrative hub for post-translational modifications (PTM) within the thin filament. There are a number of Tn phosphorylations; however, β-adrenergic-induced protein kinase A (PKA) phosphorylation of cardiac troponin I (TnI) acts as the major sarcomeric modulator of function. Traditionally, individual phosphorylation sites have been studied, yet simultaneous phosphorylation of multiple sites occurs in the heart. To fully understand the molecular mechanisms underlying cardiac muscle regulation, Tn phosphorylation must be studied as integrated events. As an initial step towards understanding Tn crosstalk, we employed a two-model phosphomimetic approach using cardiac human Tn. We studied AMP-activated protein kinase (AMPK) TnI phosphorylation (TnI-S150D) and protein kinase C (PKC) cardiac troponin T (TnT) phosphorylation (TnT-T284E) to investigate the influence of these modifications on the effects exerted by TnI PKA phosphorylation (TnI-S23/24D). The combinatorial effects of these phosphorylation events on TnI-S23/24D was determined by measuring troponin C calcium binding properties in the reconstituted thin filament. Calcium dissociated from TnI-S23/24D thin filaments at 368/s compared to 103/s for wild-type (WT). Results demonstrate that TnI-S150D alone decreases calcium dissociation by 50%, while TnI-S150D integration with TnI-S23/24D blunted TnI-S23/24D kinetics by 15%, demonstrating the intramolecular crosstalk of TnI-S150D to alter the effects of TnI PKA phosphorylation. On the other hand, TnT-T284E calcium dissociation was identical to WT, but when present with TnI-S23/24D calcium dissociation was enhanced 136% compared to TnI-S23/24D alone. This demonstrates an intermolecular influence of TnT phosphorylation on TnI PKA effects. Overall, these findings demonstrate the combinatorial effects of Tn site-specific phosphorylation by AMPK and PKC crosstalk to affect the function of TnI PKA-induced phosphorylation through both intra- and inter- molecular mechanisms. These data further highlight the importance of understanding the role of integrated Tn PTM crosstalk on cardiac contractile regulation.

Myocardial infarction in mice alters sarcomeric function via post-translational protein modification

Myocardial physiology in the aftermath of myocardial infarction (MI) before remodeling is an under-explored area of investigation. Here, we describe the effects of MI on the cardiac sarcomere with focus on the possible contributions of reactive oxygen species. We surgically induced MI in 6-7-month-old female CD1 mice by ligation of the left anterior descending coronary artery. Data were collected 3-4 days after MI or sham (SH) surgery. MI hearts demonstrated ventricular dilatation and systolic dysfunction upon echo cardiographic analysis. Sub-maximum Ca-activated tension in detergent-extracted fiber bundles from papillary muscles increased significantly in the preparations from MI hearts. Ca(2+) sensitivity increased after MI, whereas cooperativity of activation decreased. To assess myosin enzymatic integrity we measured splitting of Ca-ATP in myofibrillar preparations, which demonstrated a decline in Ca-ATPase activity of myofilament myosin. Biochemical analysis demonstrated post-translational modification of sarcomeric proteins. Phosphorylation of cardiac troponin I and myosin light chain 2 was reduced after MI in papillary samples, as measured using a phospho-specific stain. Tropomyosin was oxidized after MI, forming disulfide products detectable by diagonal non-reducing-reducing SDS-PAGE. Our analysis of myocardial protein oxidation post-MI also demonstrated increased S-glutathionylation. We functionally linked protein oxidation with sarcomere function by treating skinned fibers with the sulfhydryl reducing agent dithiothreitol, which reduced Ca(2+) sensitivity in MI, but not SH, samples. Our data indicate important structural and functional alterations to the cardiac sarcomere after MI, and the contribution of protein oxidation to this process.