Long Term Ablation of Protein Kinase A (PKA)-mediated Cardiac Troponin I Phosphorylation Leads to Excitation-Contraction Uncoupling and Diastolic Dysfunction in a Knock-in Mouse Model of Hypertrophic Cardiomyopathy

The cardiac troponin I (cTnI) R21C (cTnI-R21C) mutation has been linked to hypertrophic cardiomyopathy and renders cTnI incapable of phosphorylation by PKA in vivo. Echocardiographic imaging of homozygous knock-in mice expressing the cTnI-R21C mutation shows that they develop hypertrophy after 12 months of age and have abnormal diastolic function that is characterized by longer filling times and impaired relaxation. Electrocardiographic analyses show that older R21C mice have elevated heart rates and reduced cardiovagal tone. Cardiac myocytes isolated from older R21C mice demonstrate that in the presence of isoproterenol, significant delays in Ca²⁺ decay and sarcomere relaxation occur that are not present at 6 months of age. Although isoproterenol and stepwise increases in stimulation frequency accelerate Ca²⁺-transient and sarcomere shortening kinetics in R21C myocytes from older mice, they are unable to attain the corresponding WT values. When R21C myocytes from older mice are treated with isoproterenol, evidence of excitation-contraction uncoupling is indicated by an elevation in diastolic calcium that is frequency-dissociated and not coupled to shorter diastolic sarcomere lengths. Myocytes from older mice have smaller Ca²⁺ transient amplitudes (2.3-fold) that are associated with reductions (2.9-fold) in sarcoplasmic reticulum Ca²⁺ content. This abnormal Ca²⁺ handling within the cell may be attributed to a reduction (2.4-fold) in calsequestrin expression in conjunction with an up-regulation (1.5-fold) of Na⁺-Ca²⁺ exchanger. Incubation of permeabilized cardiac fibers from R21C mice with PKA confirmed that the mutation prevents facilitation of mechanical relaxation. Altogether, these results indicate that the inability to enhance myofilament relaxation through cTnI phosphorylation predisposes the heart to abnormal diastolic function, reduced accessibility of cardiac reserves, dysautonomia, and hypertrophy.     

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.     

Effects of phosphorylation and mutation R145G on human cardiac troponin I function.

We have studied functional consequences of the mutations R145G, S22A, and S23A of human cardiac troponin I (cTnI) and of phosphorylation of two adjacent N-terminal serine residues in the wild-type cTnI and the mutated proteins. The mutation R145G has been linked to the development of familial hypertrophic cardiomyopathy. Cardiac troponin was reconstituted from recombinant human subunits including either wild-type or mutant cTnI and was used for reconstitution of thin filaments with skeletal muscle actin and tropomyosin. The Ca(2+)-dependent thin filament-activated myosin subfragment 1 ATPase (actoS1-ATPase) activity and the in vitro motility of these filaments driven by myosin were measured as a function of the cTnI phosphorylation state. Bisphosphorylation of wild-type cTnI decreases the Ca(2+) sensitivity of the actoS1-ATPase activity and the in vitro thin filament motility by about 0.15-0.21 pCa unit. The nonconservative replacement R145G in cTnI enhances the Ca(2+) sensitivity of the actoS1-ATPase activity by about 0.6 pCa unit independent of the phosphorylation state of cTnI. Furthermore, it mimics a strong suppressing effect on both the maximum actoS1-ATPase activity and the maximum in vitro filament sliding velocity which has been observed upon bisphosphorylation of wild-type cTnI. Bisphosphorylation of the mutant cTnI-R145G itself had no such suppressing effects anymore. Differential analysis of the effect of phosphorylation of each of the two serines, Ser23 in cTnI-S22A and Ser22 in cTnI-S23A, indicates that phosphorylation of Ser23 may already be sufficient for causing the reduction of maximum actoS1-ATPase activity and thin filament sliding velocity seen upon phosphorylation of both of these serines.     

Distinct Sarcomeric Substrates Are Responsible for Protein Kinase D-mediated Regulation of Cardiac Myofilament Ca2+ Sensitivity and Cross-bridge Cycling

Protein kinase D (PKD), a serine/threonine kinase with emerging cardiovascular functions, phosphorylates cardiac troponin I (cTnI) at Ser²²/Ser²³, reduces myofilament Ca²⁺ sensitivity, and accelerates cross-bridge cycle kinetics. Whether PKD regulates cardiac myofilament function entirely through cTnI phosphorylation at Ser²²/Ser²³ remains to be established. To determine the role of cTnI phosphorylation at Ser²²/Ser²³ in PKD-mediated regulation of cardiac myofilament function, we used transgenic mice that express cTnI in which Ser²²/Ser²³ are substituted by nonphosphorylatable Ala (cTnI-Ala₂). In skinned myocardium from wild-type (WT) mice, PKD increased cTnI phosphorylation at Ser²²/Ser²³ and decreased the Ca²⁺ sensitivity of force. In contrast, PKD had no effect on the Ca²⁺ sensitivity of force in myocardium from cTnI-Ala₂ mice, in which Ser²²/Ser²³ were unavailable for phosphorylation. Surprisingly, PKD accelerated cross-bridge cycle kinetics similarly in myocardium from WT and cTnI-Ala₂ mice. Because cardiac myosin-binding protein C (cMyBP-C) phosphorylation underlies cAMP-dependent protein kinase (PKA)-mediated acceleration of cross-bridge cycle kinetics, we explored whether PKD phosphorylates cMyBP-C at its PKA sites, using recombinant C1C2 fragments with or without site-specific Ser/Ala substitutions. Kinase assays confirmed that PKA phosphorylates Ser²⁷³, Ser²⁸², and Ser³⁰², and revealed that PKD phosphorylates only Ser³⁰². Furthermore, PKD phosphorylated Ser³⁰² selectively and to a similar extent in native cMyBP-C of skinned myocardium from WT and cTnI-Ala₂ mice, and this phosphorylation occurred throughout the C-zones of sarcomeric A-bands. In conclusion, PKD reduces myofilament Ca²⁺ sensitivity through cTnI phosphorylation at Ser²²/Ser²³ but accelerates cross-bridge cycle kinetics by a distinct mechanism. PKD phosphorylates cMyBP-C at Ser³⁰², which may mediate the latter effect.     

Mutual Rescues between Two Dominant Negative Mutations in Cardiac Troponin I and Cardiac Troponin T

Troponin T (TnT) and troponin I (TnI) are two evolutionarily and functionally linked subunits of the troponin complex that regulates striated muscle contraction. We previously reported a single amino acid substitution in the highly conserved TnT-binding helix of cardiac TnI (cTnI) in wild turkey hearts in concurrence with an abnormally spliced myopathic cardiac TnT (cTnT) (Biesiadecki, B. J., Schneider, K. L., Yu, Z. B., Chong, S. M., and Jin, J. P. (2004) J. Biol. Chem. 279, 13825–13832). To investigate the functional effect of this cTnI mutation and its potential value in compensating for the cTnT abnormality, we developed transgenic mice expressing the mutant cTnI (K118C) in the heart with or without the deletion of the endogenous cTnI gene to mimic the homozygote and heterozygote of wild turkeys. Double and triple transgenic mice were created by crossing the cTnI-K118C lines with transgenic mice overexpressing the myopathic cTnT (exon 7 deletion). Functional studies of ex vivo working hearts found that cTnI-K118C alone had a dominantly negative effect on diastolic function and blunted the inotropic responses of cardiac muscle to β-adrenergic stimuli without abolishing the protein kinase A-dependent phosphorylation of cTnI. When co-expressed with the cTnT mutation, cTnI-K118C corrected the significant depression of systolic function caused by cTnT exon 7 deletion, and the co-existence of exon 7-deleted cTnT minimized the diastolic abnormality of cTnI-K118C. Characterization of this naturally selected pair of mutually rescuing mutations demonstrated that TnI-TnT interaction is a critical link in the Ca²⁺ signaling and β-adrenergic regulation in cardiac muscle, suggesting a potential target for the treatment of troponin cardiomyopathies and heart failure.     

Phosphorylation-dependent conformational transition of the cardiac specific N-extension of troponin I in cardiac troponin

We present here the solution structure for the bisphosphorylated form of the cardiac N-extension of troponin I (cTnI(1-32)), a region for which there are no previous high-resolution data. Using this structure, the X-ray crystal structure of the cardiac troponin core, and uniform density models of the troponin components derived from neutron contrast variation data, we built atomic models for troponin that show the conformational transition in cardiac troponin induced by bisphosphorylation. In the absence of phosphorylation, our NMR data and sequence analyses indicate a less structured cardiac N-extension with a propensity for a helical region surrounding the phosphorylation motif, followed by a helical C-terminal region (residues 25-30). In this conformation, TnI(1-32) interacts with the N-lobe of cardiac troponin C (cTnC) and thus is positioned to modulate myofilament Ca2+-sensitivity. Bisphosphorylation at Ser23/24 extends the C-terminal helix (residues 21-30) which results in weakening interactions with the N-lobe of cTnC and a re-positioning of the acidic amino terminus of cTnI(1-32) for favorable interactions with basic regions, likely the inhibitory region of cTnI. An extended poly(L-proline)II helix between residues 11 and 19 serves as the rigid linker that aids in re-positioning the amino terminus of cTnI(1-32) upon bisphosphorylation at Ser23/24. We propose that it is these electrostatic interactions between the acidic amino terminus of cTnI(1-32) and the basic inhibitory region of troponin I that induces a bending of cTnI at the end that interacts with cTnC. This model provides a molecular mechanism for the observed changes in cross-bridge kinetics upon TnI phosphorylation.     

Unraveling molecular complexity of phosphorylated human cardiac troponin I by top down electron capture dissociation/electron transfer dissociation mass spectrometry

Cardiac troponin I (cTnI), the inhibitory subunit of the thin filament troponin-tropomyosin regulatory complex, is required for heart muscle relaxation during the cardiac cycle. Expressed only in cardiac muscle, cTnI is widely used in the clinic as a serum biomarker of cardiac injury. In vivo function of cTnI is influenced by phosphorylation and proteolysis; therefore analysis of post-translational modifications of the intact protein should greatly facilitate the understanding of cardiac regulatory mechanisms and may improve cTnI as a disease biomarker. cTnI (24 kDa, pI approximately 9.5) contains twelve serine, eight threonine, and three tyrosine residues, which presents a challenge for unequivocal identification of phosphorylation sites and quantification of positional isomers. In this study, we used top down electron capture dissociation and electron transfer dissociation MS to unravel the molecular complexity of cTnI purified from human heart tissue. High resolution MS spectra of human cTnI revealed a high degree of heterogeneity, corresponding to phosphorylation, acetylation, oxidation, and C-terminal proteolysis. Thirty-six molecular ions of cTnI were detected in a single ESI/FTMS spectrum despite running as a single sharp band on SDS-PAGE. Electron capture dissociation of monophosphorylated cTnI localized two major basal phosphorylation sites: a well known site at Ser(22) and a novel site at Ser(76)/Thr(77), each with partial occupancy (Ser(22): 53%; Ser(76)/Thr(77): 36%). Top down MS(3) analysis of diphosphorylated cTnI revealed occupancy of Ser(23) only in diphosphorylated species consistent with sequential (or ordered) phosphorylation/dephosphorylation of the Ser(22/23) pair. Top down MS of cTnI provides unique opportunities for unraveling its molecular complexity and for quantification of phosphorylated positional isomers thus allowing establishment of the relevance of such modifications to physiological functions and disease status.     

Troponin I serines 43/45 and regulation of cardiac myofilament function

We studied Ca(2+) dependence of tension and actomyosin ATPase rate in detergent extracted fiber bundles isolated from transgenic mice (TG), in which cardiac troponin I (cTnI) serines 43 and 45 were mutated to alanines (cTnI S43A/S45A). Basal phosphorylation levels of cTnI were lower in TG than in wild-type (WT) mice, but phosphorylation of cardiac troponin T was increased. Compared with WT, TG fiber bundles showed a 13% decrease in maximum tension and a 20% increase in maximum MgATPase activity, yielding an increase in tension cost. Protein kinase C (PKC) activation with endothelin (ET) or phenylephrine plus propranolol (PP) before detergent extraction induced a decrease in maximum tension and MgATPase activity in WT fibers, whereas ET or PP increased maximum tension and stiffness in TG fibers. TG MgATPase activity was unchanged by ET but increased by PP. Measurement of protein phosphorylation revealed differential effects of agonists between WT and TG myofilaments and within the TG myofilaments. Our results demonstrate the importance of PKC-mediated phosphorylation of cTnI S43/S45 in the control of myofilament activation and cross-bridge cycling rate.     

In vivo and in vitro analysis of cardiac troponin I phosphorylation

Adrenergic stimulation induces positive changes in cardiac contractility and relaxation. Cardiac troponin I is phosphorylated at different sites by protein kinase A and protein kinase C, but the effects of these post-translational modifications on the rate and extent of contractility and relaxation during beta-adrenergic stimulation in the intact animal remain obscure. To investigate the effect(s) of complete and chronic cTnI phosphorylation on cardiac function, we generated transgenic animals in which the five possible phosphorylation sites were replaced with aspartic acid, mimicking a constant state of complete phosphorylation (cTnI-AllP). We hypothesized that chronic and complete phosphorylation of cTnI might result in increased morbidity or mortality, but complete replacement with the transgenic protein was benign with no detectable pathology. To differentiate the effects of the different phosphorylation sites, we generated another mouse model, cTnI-PP, in which only the protein kinase A phosphorylation sites (Ser(23)/Ser(24)) were mutated to aspartic acid. In contrast to the cTnIAllP, the cTnI-PP mice showed enhanced diastolic function under basal conditions. The cTnI-PP animals also showed augmented relaxation and contraction at higher heart rates compared with the nontransgenic controls. Nuclear magnetic resonance amide proton/nitrogen chemical shift analysis of cardiac troponin C showed that, in the presence of cTnI-AllP and cTnI-PP, the N terminus exhibits a more closed conformation, respectively. The data show that protein kinase C phosphorylation of cTnI plays a dominant role in depressing contractility and exerts an antithetic role on the ability of protein kinase A to increase relaxation.     

Protein kinase C zeta. A novel regulator of both phosphorylation and de-phosphorylation of cardiac sarcomeric proteins

Our experiments investigated associations of specific isoforms of protein kinase C (PKC) with individual proteins in the cardiac troponin complex. Troponin I (cTnI) associated with PKCepsilon and zeta and troponin T (cTnT) associated with PKC alpha, delta, and epsilon. Based on its association with cTnI, we hypothesized that PKCzeta is a major regulator of myofilament protein phosphorylation. To test this, we infected adult cardiac myocytes with adenoviral constructs containing DsRed monomer-tagged wild type (WT) and the following constitutively active forms of PKCzeta: the pseudo-substrate region (A119E), 3'-phospho-inositide-dependent kinase-1 (T410E), and auto-phosphorylation (T560E). The A119E and T410E mutants displayed increased localization to the Z-discs compared with WT and T560E. Immunoprecipitations were performed in myocytes expressing PKCzeta using PKC phospho-motif antibodies to determine the phosphorylation of cTnI, cTnT, tropomyosin, myosin-binding protein C, and desmin. We did not find serine (Ser) phosphorylation of cTnI or cTnT. However, we observed a significant decrease in threonine (Thr) phosphorylation of cTnI and cTnT notably by PKCzeta T560E. Ser phosphorylation of tropomyosin was increased by all three active mutants of PKCzeta. Ser/Thr phosphorylation of myosin-binding protein C increased primarily by PKCzeta A119E. Both PKCzeta A119E and T410E mutants increased desmin Ser/Thr phosphorylation. To explain the apparent Thr dephosphorylation of cTnI and cTnT, we hypothesized that PKCzeta exists as a complex with p21-activated kinase-1 (Pak1) and protein phosphatase 2A (PP2A), and this was confirmed by immunoprecipitation Western blot. Our data demonstrate that PKCzeta is a novel regulator of myofilament protein phosphorylation.     

Troponin I Mutations R146G and R21C Alter Cardiac Troponin Function,Contractile Properties,and Modulation by Protein Kinase A (PKA)-mediated Phosphorylation

Two hypertrophic cardiomyopathy-associated cardiac troponin I (cTnI) mutations, R146G and R21C, are located in different regions of cTnI, the inhibitory peptide and the cardiac-specific N terminus. We recently reported that these regions may interact when Ser-23/Ser-24 are phosphorylated, weakening the interaction of cTnI with cardiac TnC. Little is known about how these mutations influence the affinity of cardiac TnC for cTnI (K_C-I) or contractile kinetics during β-adrenergic stimulation. Here, we tested how cTnI^R146G or cTnI^R21C influences contractile activation and relaxation and their response to protein kinase A (PKA). Both mutations significantly increased Ca²⁺ binding affinity to cTn (K_Ca) and K_C-I. PKA phosphorylation resulted in a similar reduction of K_Ca for all complexes, but K_C-I was reduced only with cTnI^WT. cTnI^WT, cTnI^R146G, and cTnI^R21C were complexed into cardiac troponin and exchanged into rat ventricular myofibrils, and contraction/relaxation kinetics were measured ± PKA phosphorylation. Maximal tension (T_max) was maintained for cTnI^R146G- and cTnI^R21C-exchanged myofibrils, and Ca²⁺ sensitivity of tension (pCa₅₀) was increased. PKA phosphorylation decreased pCa₅₀ for cTnI^WT-exchanged myofibrils but not for either mutation. PKA phosphorylation accelerated the early slow phase relaxation for cTnI^WT myofibrils, especially at Ca²⁺ levels that the heart operates in vivo. Importantly, this effect was blunted for cTnI^R146G- and cTnI^R21C-exchanged myofibrils. Molecular dynamics simulations suggest both mutations inhibit formation of intra-subunit contacts between the N terminus and the inhibitory peptide of cTnI that is normally seen with WT-cTn upon PKA phosphorylation. Together, our results suggest that cTnI^R146G and cTnI^R21C blunt PKA modulation of activation and relaxation kinetics by prohibiting cardiac-specific N-terminal interaction with the cTnI inhibitory peptide.     

Tyrosine phosphorylation modifies protein kinase C delta-dependent phosphorylation of cardiac troponin I

Our study identifies tyrosine phosphorylation as a novel protein kinase Cdelta (PKCdelta) activation mechanism that modifies PKCdelta-dependent phosphorylation of cardiac troponin I (cTnI), a myofilament regulatory protein. PKCdelta phosphorylates cTnI at Ser23/Ser24 when activated by lipid cofactors; Src phosphorylates PKCdelta at Tyr311 and Tyr332 leading to enhanced PKCdelta autophosphorylation at Thr505 (its activation loop) and PKCdelta-dependent cTnI phosphorylation at both Ser23/Ser24 and Thr144. The Src-dependent acquisition of cTnI-Thr144 kinase activity is abrogated by Y311F or T505A substitutions. Treatment of detergent-extracted single cardiomyocytes with lipid-activated PKCdelta induces depressed tension at submaximum but not maximum [Ca2+] as expected for cTnI-Ser23/Ser24 phosphorylation. Treatment of myocytes with Src-activated PKCdelta leads to depressed maximum tension and cross-bridge kinetics, attributable to a dominant effect of cTnI-Thr144 phosphorylation. Our data implicate PKCdelta-Tyr311/Thr505 phosphorylation as dynamically regulated modifications that alter PKCdelta enzymology and allow for stimulus-specific control of cardiac mechanics during growth factor stimulation and oxidative stress.     

A Mutation in TNNC1-encoded Cardiac Troponin C, TNNC1-A31S, Predisposes to Hypertrophic Cardiomyopathy and Ventricular Fibrillation

Defined as clinically unexplained hypertrophy of the left ventricle, hypertrophic cardiomyopathy (HCM) is traditionally understood as a disease of the cardiac sarcomere. Mutations in TNNC1-encoded cardiac troponin C (cTnC) are a relatively rare cause of HCM. Here, we report clinical and functional characterization of a novel TNNC1 mutation, A31S, identified in a pediatric HCM proband with multiple episodes of ventricular fibrillation and aborted sudden cardiac death. Diagnosed at age 5, the proband is family history-negative for HCM or sudden cardiac death, suggesting a de novo mutation. TnC-extracted cardiac skinned fibers were reconstituted with the cTnC-A31S mutant, which increased Ca(2+) sensitivity with no effect on the maximal contractile force generation. Reconstituted actomyosin ATPase assays with 50% cTnC-A31S:50% cTnC-WT demonstrated Ca(2+) sensitivity that was intermediate between 100% cTnC-A31S and 100% cTnC-WT, whereas the mutant increased the activation of the actomyosin ATPase without affecting the inhibitory qualities of the ATPase. The secondary structure of the cTnC mutant was evaluated by circular dichroism, which did not indicate global changes in structure. Fluorescence studies demonstrated increased Ca(2+) affinity in isolated cTnC, the troponin complex, thin filament, and to a lesser degree, thin filament with myosin subfragment 1. These results suggest that this mutation has a direct effect on the Ca(2+) sensitivity of the myofilament, which may alter Ca(2+) handling and contribute to the arrhythmogenesis observed in the proband. In summary, we report a novel mutation in the TNNC1 gene that is associated with HCM pathogenesis and may predispose to the pathogenesis of a fatal arrhythmogenic subtype of HCM.     

Protein kinase A-induced myofilament desensitization to Ca(2+) as a result of phosphorylation of cardiac myosin-binding protein C

In skinned myocardium, cyclic AMP-dependent protein kinase A (PKA)-catalyzed phosphorylation of cardiac myosin-binding protein C (cMyBP-C) and cardiac troponin I (cTnI) is associated with a reduction in the Ca(2+) responsiveness of myofilaments and an acceleration in the kinetics of cross-bridge cycling, although the respective contribution of these two proteins remains controversial. To further examine the relative roles that cTnI and cMyBP-C phosphorylation play in altering myocardial function, we determined the Ca(2+) sensitivity of force (pCa(50)) and the activation dependence of the rate of force redevelopment (k(tr)) in control and PKA-treated mouse myocardium (isolated in the presence of 2,3-butanedione monoxime) expressing: (a) phosphorylatable cTnI and cMyBP-C (wild type [WT]), (b) phosphorylatable cTnI on a cMyBP-C-null background (cMyBP-C(-/-)), (c) nonphosphorylatable cTnI with serines(23/24/43/45) and threonine(144) mutated to alanines (cTnI(Ala5)), and (d) nonphosphorylatable cTnI on a cMyBP-C-null background (cTnI(Ala5)/cMyBP-C(-/-)). Here, PKA treatment decreased pCa(50) in WT, cTnI(Ala5), and cMyBP-C(-/-) myocardium by 0.13, 0.08, and 0.09 pCa units, respectively, but had no effect in cTnI(Ala5)/cMyBP-C(-/-) myocardium. In WT and cTnI(Ala5) myocardium, PKA treatment also increased k(tr) at submaximal levels of activation; however, PKA treatment did not have an effect on k(tr) in cMyBP-C(-/-) or cTnI(Ala5)/cMyBP-C(-/-) myocardium. In addition, reconstitution of cTnI(Ala5)/cMyBP-C(-/-) myocardium with recombinant cMyBP-C restored the effects of PKA treatment on pCa(50) and k(tr) reported in cTnI(Ala5) myocardium. Collectively, these results indicate that the attenuation in myofilament force response to PKA occurs as a result of both cTnI and cMyBP-C phosphorylation, and that the reduction in pCa(50) mediated by cMyBP-C phosphorylation most likely arises from an accelerated cross-bridge cycling kinetics partly as a result of an increased rate constant of cross-bridge detachment.     

Drastic Ca2+ sensitization of myofilament associated with a small structural change in troponin I in inherited restrictive cardiomyopathy

Six missense mutations in human cardiac troponin I (cTnI) were recently found to cause restrictive cardiomyopathy (RCM). We have bacterially expressed and purified these human cTnI mutants and examined their functional and structural consequences. Inserting the human cTnI into skinned cardiac muscle fibers showed that these mutations had much greater Ca2+-sensitizing effects on force generation than the cTnI mutations in hypertrophic cardiomyopathy (HCM). The mutation K178E in the second actin-tropomyosin (Tm) binding region showed a particularly potent Ca2+-sensitizing effect among the six RCM-causing mutations. Circular dichroism and nuclear magnetic resonance spectroscopy revealed that this mutation does not extensively affect the structure of the whole cTnI molecule, but induces an unexpectedly subtle change in the structure of a region around the mutated residue. The results indicate that the K178E mutation has a localized effect on a structure that is critical to the regulatory function of the second actin-Tm binding region of cTnI. The present study also suggests that both HCM and RCM involving cTnI mutations share a common feature of increased Ca2+ sensitivity of cardiac myofilament, but more severe change in Ca2+ sensitivity is associated with the clinical phenotype of RCM.     

Removal of the Cardiac Troponin I N-terminal Extension Improves Cardiac Function in Aged Mice

The cardiac troponin I (cTnI) isoform contains a unique N-terminal extension that functions to modulate activation of cardiac myofilaments. During cardiac remodeling restricted proteolysis of cTnI removes this cardiac specific N-terminal modulatory extension to alter myofilament regulation. We have demonstrated expression of the N-terminal-deleted cTnI (cTnI-ND) in the heart decreased the development of the cardiomyopathy like phenotype in a β-adrenergic-deficient transgenic mouse model. To investigate the potential beneficial effects of cTnI-ND on the development of naturally occurring cardiac dysfunction, we measured the hemodynamic and biochemical effects of cTnI-ND transgenic expression in the aged heart. Echocardiographic measurements demonstrate cTnI-ND transgenic mice exhibit increased systolic and diastolic functions at 16 months of age compared with age-matched controls. This improvement likely results from decreased Ca²⁺ sensitivity and increased cross-bridge kinetics as observed in skinned papillary bundles from young transgenic mice prior to the effects of aging. Hearts of cTnI-ND transgenic mice further exhibited decreased β myosin heavy chain expression compared to age matched non-transgenic mice as well as altered cTnI phosphorylation. Finally, we demonstrated cTnI-ND expressed in the heart is not phosphorylated indicating the cTnI N-terminal is necessary for the higher level phosphorylation of cTnI. Taken together, our data suggest the regulated proteolysis of cTnI during cardiac stress to remove the unique cardiac N-terminal extension functions to improve cardiac contractility at the myofilament level and improve overall cardiac function.     

Protein kinase D isoforms are activated in an agonist-specific manner in cardiomyocytes

Protein kinase D (PKD) exists as a family of structurally related enzymes that are activated through similar phosphorylation-dependent mechanisms involving protein kinase C (PKC). While individual PKD isoforms could in theory mediate distinct biological functions, previous studies identify a high level of functional redundancy for PKD1 and PKD2 in various cellular contexts. This study shows that PKD1 and PKD2 are activated in a stimulus-specific manner in neonatal cardiomyocytes. The α(1)-adrenergic receptor agonist norepinephrine selectively activates PKD1, thrombin and PDGF selectively activate PKD2, and endothelin-1 and PMA activate both PKD1 and PKD2. PKC activity is implicated in the α(1)-adrenergic receptor pathway that activates PKD1 and the thrombin- and PDGF-dependent pathways that activate PKD2. Endothelin-1 activates PKD via both rapid PKC-dependent and more sustained PKC-independent mechanisms. The functional consequences of PKD activation were assessed by tracking phosphorylation of CREB and cardiac troponin I (cTnI), two physiologically relevant PKD substrates in cardiomyocytes. We show that overexpression of an activated PKD1-S744E/S748E transgene increases CREB-Ser(133) and cTnI-Ser(23)/Ser(24) phosphorylation, but agonist-dependent pathways that activate native PKD1 or PKD2 selectively increase CREB-Ser(133) phosphorylation; there is no associated increase in cTnI-Ser(23)/Ser(24) phosphorylation. Gene silencing studies provide unanticipated evidence that PKD1 down-regulation leads to a compensatory increase in PKD2 activity and that down-regulation of PKD1 (alone or in combination with PKD2) leads to an increase in CREB-Ser(133) phosphorylation. Collectively, these studies identify distinct roles for native PKD1 and PKD2 enzymes in stress-dependent pathways that influence cardiac remodeling and the progression of heart failure.     

Altered regulatory properties of human cardiac troponin I mutants that cause hypertrophic cardiomyopathy

Cardiac troponin I (cTnI) is the inhibitory component of the troponin complex and is involved in the calcium control of heart muscle contraction. Recently, specific missense mutations of the cTnI gene (TNNI3) have been shown to cause familial hypertrophic cardiomyopathy (HCM). We have analyzed the functional effects of two HCM mutations (R145G and R162W) using purified recombinant cTnI. Both mutations gave reduced inhibition of actin-tropomyosin-activated myosin ATPase, both in experiments using cTnI alone and in those using reconstituted human cardiac troponin under relaxing conditions. Both mutant troponin complexes also conferred increased calcium sensitivity of ATPase regulation. Studies on wild type/R145G mutant mixtures showed that the wild type phenotype was dominant in that the inhibition and the calcium sensitivity conferred by a 50:50 mixture was more similar to wild type than expected. Surface plasmon resonance-based assays showed that R162W mutant had an increased affinity for troponin C in the presence of calcium. This observation may contribute to the increased calcium sensitivity found with this mutant and also corroborates recent structural data. The observed decreased inhibition and increased calcium sensitivity suggest that these mutations may cause HCM via impaired relaxation rather than the impaired contraction seen with some other classes of HCM mutants.     

alpha-Adrenergic response and myofilament activity in mouse hearts lacking PKC phosphorylation sites on cardiac TnI

Protein kinase C (PKC)-mediated phosphorylation of cardiac myofilament (MF) proteins has been shown to depress the actomyosin interaction and may be important during heart failure. Biochemical studies indicate that phosphorylation of Ser(43) and Ser(45) of cardiac troponin I (cTnI) plays a substantial role in the PKC-mediated depression. We studied intact and detergent-extracted papillary muscles from nontransgenic (NTG) and transgenic (TG) mouse hearts that express a mutant cTnI (Ser43Ala, Ser45Ala) that lacks specific PKC-dependent phosphorylation sites. Treatment of NTG papillary muscles with phenylephrine (PE) resulted in a transient increase and a subsequent 62% reduction in peak twitch force. TG muscles showed no transient increase and only a 45% reduction in force. There was a similar difference in maximum tension between NTG and TG fiber bundles that had been treated with a phorbol ester and had received subsequent detergent extraction. Although levels of cTnI phosphorylation correlated with these differences, the TG fibers also demonstrated a decrease in phosphorylation of cardiac troponin T. The PKC-specific inhibitor chelerythrine inhibited these responses. Our data provide evidence that specific PKC-mediated phosphorylation of Ser(43) and Ser(45) of cTnI plays an important role in regulating force development in the intact myocardium.