AMP-activated protein kinase phosphorylates cardiac troponin I and alters contractility of murine ventricular myocytes.

RATIONALE: AMP-activated protein kinase (AMPK) is an important regulator of energy balance and signaling in the heart. Mutations affecting the regulatory γ2 subunit have been shown to cause an essentially cardiac-restricted phenotype of hypertrophy and conduction disease, suggesting a specific role for this subunit in the heart. OBJECTIVE: The γ isoforms are highly conserved at their C-termini but have unique N-terminal sequences, and we hypothesized that the N-terminus of γ2 may be involved in conferring substrate specificity or in determining intracellular localization. METHODS AND RESULTS: A yeast 2-hybrid screen of a human heart cDNA library using the N-terminal 273 residues of γ2 as bait identified cardiac troponin I (cTnI) as a putative interactor. In vitro studies showed that cTnI is a good AMPK substrate and that Ser150 is the principal residue phosphorylated. Furthermore, on AMPK activation during ischemia, Ser150 is phosphorylated in whole hearts. Using phosphomimics, measurements of actomyosin ATPase in vitro and force generation in demembraneated trabeculae showed that modification at Ser150 resulted in increased Ca(2+) sensitivity of contractile regulation. Treatment of cardiomyocytes with the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) resulted in increased myocyte contractility without changing the amplitude of Ca(2+) transient and prolonged relaxation despite shortening the time constant of Ca(2+) transient decay (tau). Compound C prevented the effect of AICAR on myocyte function. These results suggest that AMPK activation increases myocyte contraction and prolongs relaxation by increasing myofilament Ca(2+) sensitivity. CONCLUSIONS: We conclude that cTnI phosphorylation by AMPK may represent a novel mechanism of regulation of cardiac function.

Generation and functional characterization of knock-in mice harboring the cardiac troponin I-R21C mutation associated with hypertrophic cardiomyopathy.

The R21C substitution in cardiac troponin I (cTnI) is the only identified mutation within its unique N-terminal extension that is associated with hypertrophic cardiomyopathy (HCM) in man. Particularly, this mutation is located in the consensus sequence for ß-adrenergic-activated protein kinase A (PKA)-mediated phosphorylation. The mechanisms by which this mutation leads to heart disease are still unclear. Therefore, we generated cTnI knock-in mouse models carrying an R21C mutation to evaluate the resultant functional consequences. Measuring the in vivo levels of incorporated mutant and WT cTnI, and their basal phosphorylation levels by top-down mass spectrometry demonstrated: 1) a dominant-negative effect such that, the R21C+/- hearts incorporated 24.9% of the mutant cTnI within the myofilament; and 2) the R21C mutation abolished the in vivo phosphorylation of Ser(23)/Ser(24) in the mutant cTnI. Adult heterozygous (R21C+/-) and homozygous (R21C+/+) mutant mice activated the fetal gene program and developed a remarkable degree of cardiac hypertrophy and fibrosis. Investigation of cardiac skinned fibers isolated from WT and heterozygous mice revealed that the WT cTnI was completely phosphorylated at Ser(23)/Ser(24) unless the mice were pre-treated with propranolol. After propranolol treatment (-PKA), the pCa-tension relationships of all three mice (i.e. WT, R21C+/-, and R21C+/+) were essentially the same. However, after treatment with propranolol and PKA, the R21C cTnI mutation reduced (R21C+/-) or abolished (R21C+/+) the well known decrease in the Ca(2+) sensitivity of tension that accompanies Ser(23)/Ser(24) cTnI phosphorylation. Altogether, the combined effects of the R21C mutation appear to contribute toward the development of HCM and suggest that another physiological role for the phosphorylation of Ser(23)/Ser(24) in cTnI is to prevent cardiac hypertrophy.

Sexually dimorphic myofilament function and cardiac troponin I phosphospecies distribution in hypertrophic cardiomyopathy mice.

The pathological progression of hypertrophic cardiomyopathy (HCM) is sexually dimorphic such that male HCM mice develop phenotypic indicators of cardiac disease well before female HCM mice. Here, we hypothesized that alterations in myofilament function underlies, in part, this sex dimorphism in HCM disease development. Firstly, 10-12month female HCM (harboring a mutant [R403Q] myosin heavy chain) mice presented with proportionately larger hearts than male HCM mice. Next, we determined Ca(2+)-sensitive tension development in demembranated cardiac trabeculae excised from 10-12month female and male HCM mice. Whereas HCM did not impact Ca(2+)-sensitive tension development in male trabeculae, female HCM trabeculae were more sensitive to Ca(2+) than wild-type (WT) counterparts and both WT and HCM males. We hypothesized that the underlying cause of this sex difference in Ca(2+)-sensitive tension development was due to changes in Ca(2+) handling and sarcomeric proteins, including expression of SR Ca(2+) ATPase (2a) (SERCA2a), ß-myosin heavy chain (ß-MyHC) and post-translational modifications of myofilament proteins. Female HCM hearts showed an elevation of SERCA2a and ß-MyHC protein whereas male HCM hearts showed a similar elevation of ß-MyHC protein but a reduced level of cardiac troponin T (cTnT) phosphorylation. We also measured the distribution of cardiac troponin I (cTnI) phosphospecies using phosphate-affinity SDS-PAGE. The distribution of cTnI phosphospecies depended on sex and HCM. In conclusion, female and male HCM mice display sex dimorphic myofilament function that is accompanied by a sex- and HCM-dependent distribution of sarcomeric proteins and cTnI phosphospecies.

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 Ser(22)/Ser(23), reduces myofilament Ca(2+) sensitivity, and accelerates cross-bridge cycle kinetics. Whether PKD regulates cardiac myofilament function entirely through cTnI phosphorylation at Ser(22)/Ser(23) remains to be established. To determine the role of cTnI phosphorylation at Ser(22)/Ser(23) in PKD-mediated regulation of cardiac myofilament function, we used transgenic mice that express cTnI in which Ser(22)/Ser(23) are substituted by nonphosphorylatable Ala (cTnI-Ala(2)). In skinned myocardium from wild-type (WT) mice, PKD increased cTnI phosphorylation at Ser(22)/Ser(23) and decreased the Ca(2+) sensitivity of force. In contrast, PKD had no effect on the Ca(2+) sensitivity of force in myocardium from cTnI-Ala(2) mice, in which Ser(22)/Ser(23) were unavailable for phosphorylation. Surprisingly, PKD accelerated cross-bridge cycle kinetics similarly in myocardium from WT and cTnI-Ala(2) 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 Ser(273), Ser(282), and Ser(302), and revealed that PKD phosphorylates only Ser(302). Furthermore, PKD phosphorylated Ser(302) selectively and to a similar extent in native cMyBP-C of skinned myocardium from WT and cTnI-Ala(2) mice, and this phosphorylation occurred throughout the C-zones of sarcomeric A-bands. In conclusion, PKD reduces myofilament Ca(2+) sensitivity through cTnI phosphorylation at Ser(22)/Ser(23) but accelerates cross-bridge cycle kinetics by a distinct mechanism. PKD phosphorylates cMyBP-C at Ser(302), which may mediate the latter effect.

Chronic rhinosinusitis with nasal polyps: a proteomic analysis.

OBJECTIVES: Chronic rhinosinusitis with nasal polyps (CRSwNP) is a severe subtype of chronic rhinosinusitis that can affect patients despite medical and surgical interventions. The purpose of this study was to utilize the techniques of proteomics to investigate differences in protein abundance within the sinonasal mucosa of patients with CRSwNP compared to healthy controls. METHODS: In a case-control study at a tertiary-care academic medical center, sinonasal mucosa was harvested from 3 patients with CRSwNP and 3 control patients undergoing transsphenoidal excision of pituitary tumors. Two-dimensional gel electrophoresis was used to identify proteins with elevated or reduced abundance in CRSwNP patients compared to controls. The proteins showing the greatest abundance differences were characterized by mass spectrometry. RESULTS: More than 300 differentially abundant proteins (p < or = 0.05) were identified. Many of these protein species were involved in the host inflammatory response. Proteins up-regulated in CRSwNP patients included eosinophil lysophospholipase by a ratio (R) of 18.13, RHO-GDP dissociation inhibitor 2 (R = 2.80), and apolipoprotein A-1 (R = 1.73). Down-regulated proteins in CRSwNP patients included catalase (R = -5.87), annexin A1 (R = -6.27), and keratin II-8 (R = -6.73). A detailed analysis of additional protein species is outlined. CONCLUSIONS: The proteomic approach allows detection of significant differences in protein abundance in CRSwNP and provides unique insight into the pathophysiology of this common disease.

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.

In vivo phosphorylation site mapping in mouse cardiac troponin I by high resolution top-down electron capture dissociation mass spectrometry: Ser22/23 are the only sites basally phosphorylated.

Cardiac troponin I (cTnI) is the inhibitory subunit of cardiac troponin, a key myofilament regulatory protein complex located on the thin filaments of the contractile apparatus. cTnI is uniquely specific for the heart and is widely used in clinics as a serum biomarker for cardiac injury. Phosphorylation of cTnI plays a critical role in modulating cardiac function. cTnI is known to be regulated by protein kinase A and protein kinase C at five sites, Ser22/Ser23, Ser42/44, and Thr143, primarily based on results from in vitro phosphorylation assays by the specific kinase(s). However, a comprehensive characterization of phosphorylation of mouse cTnI occurring in vivo has been lacking. Herein, we have employed top-down mass spectrometry (MS) methodology with electron capture dissociation for precise mapping of in vivo phosphorylation sites of cTnI affinity purified from wild-type and transgenic mouse hearts. As demonstrated, top-down MS (analysis of intact proteins) is an extremely valuable technology for global characterization of labile phosphorylation occurring in vivo without a priori knowledge. Our top-down MS data unambiguously identified Ser22/23 as the only two sites basally phosphorylated in wild-type mouse cTnI with full sequence coverage, which was confirmed by the lack of phosphorylation in cTnI-Ala(2) transgenic mice where Ser22/23 in cTnI have been rendered nonphosphorylatable by mutation to alanine.

Differential roles of cardiac myosin-binding protein C and cardiac troponin I in the myofibrillar force responses to protein kinase A phosphorylation.

The heart is remarkably adaptable in its ability to vary its function to meet the changing demands of the circulatory system. During times of physiological stress, cardiac output increases in response to increased sympathetic activity, which results in protein kinase A (PKA)-mediated phosphorylations of the myofilament proteins cardiac troponin (cTn)I and cardiac myosin-binding protein (cMyBP)-C. Despite the importance of this mechanism, little is known about the relative contributions of cTnI and cMyBP-C phosphorylation to increased cardiac contractility. Using engineered mouse lines either lacking cMyBP-C (cMyBP-C(-/-)) or expressing a non-PKA phosphorylatable cTnI (cTnI(ala2)), or both (cMyBP-C(-/-)/cTnI(ala2)), we investigated the roles of cTnI and cMyBP-C phosphorylation in the regulation of the stretch-activation response. PKA treatment of wild-type and cTnI(ala2) skinned ventricular myocardium accelerated stretch activation such that the response was indistinguishable from stretch activation of cMyBP-C(-/-) or cMyBP-C(-/-)/cTnI(ala2) myocardium; however, PKA had no effect on stretch activation in cMyBP-C(-/-) or cMyBP-C(-/-)/cTnI(ala2) myocardium. These results indicate that the acceleration of stretch activation in wild-type and cTnI(ala2) myocardium is caused by phosphorylation of cMyBP-C and not cTnI. We conclude that the primary effect of PKA phosphorylation of cTnI is reduced Ca(2+) sensitivity of force, whereas phosphorylation of cMyBP-C accelerates the kinetics of force development. These results predict that PKA phosphorylation of myofibrillar proteins in living myocardium contributes to accelerated relaxation in diastole and increased rates of force development in systole.

Endothelin receptor dimers evaluated by FRET, ligand binding, and calcium mobilization.

Endothelin-1 (ET-1) mediates physiological responses via endothelin A (ET(A)) and B (ET(B)) receptors, which may form homo- and heterodimers with unknown function. Here, we investigated ET-receptor dimerization using fluorescence resonance energy transfer (FRET) between receptors tagged with CFP (donor) and receptors tagged with tetracysteine-FlAsH (fluorescein arsenical hairpin) (acceptor) expressed in HEK293 cells. FRET efficiencies were 15%, 22%, and 27% for ET(A)/ET(A), ET(B)/ET(B), and ET(A)/ET(B), respectively, and dimerization was further supported by coimmunoprecipitation. For all dimer pairs, the natural but nonselective ligand ET-1 rapidly (50%, but did not detectably reduce coimmunoprecipitation. ET-1 stimulated a transient increase in intracellular Ca(2+) ([Ca(2+)](i)) lasting 1-2 min for both homodimer pairs, and these ET-1 actions on FRET and [Ca(2+)](i) elevation were blocked by the appropriate subtype-selective antagonist. In contrast, ET(A)/ET(B) heterodimers mediated a sustained [Ca(2+)](i) increase lasting >10 min, and required a combination of ET(A) and ET(B) antagonists to block the observed FRET and [Ca(2+)](i) responses. The sensitive CFP/FlAsH FRET assay used here provides new insights into endothelin-receptor dimer function, and represents a unique approach to characterize G-protein-coupled receptor oligomers, including their pharmacology.

Targeting of endothelin receptor-B to the neural crest.

Endothelin receptor B (Ednrb) plays a critical role in the development of melanocytes and neurons and glia of the enteric nervous system. These distinct neural crest-derived cell types express Ednrb and share the property of intercalating into tissues, such as the intestine whose muscle precursor cells also express Ednrb. Such widespread Ednrb expression has been a significant obstacle in establishing precise roles for Ednrb in development. We describe here the production of an Ednrb allele floxed at exon 3 and its use in excising the receptor from mouse neural crest cells by use of Cre-recombinase driven by the Wnt1 promoter. Mice born with neural crest-specific excision of Ednrb possess aganglionic colon, lack trunk pigmentation, and die within 5 weeks due to megacolon. Ednrb receptor expression in these animals is absent only in the neural crest but present in surrounding smooth muscle cells. The absence of Ednrb from crest cells also results in a compensatory upregulation of Ednrb expression in other cells within the gut. We conclude that Ednrb loss only in neural crest cells is sufficient to produce the Hirschsprungs disease phenotype observed with genomic Ednrb mutations.

Single amino acid sequence polymorphisms in rat cardiac troponin revealed by top-down tandem mass spectrometry.

Heterotrimeric cardiac troponin (cTn) is a critical component of the thin filament regulatory complex in cardiac muscle. Two of the three subunits, cTnI and cTnT, are subject to post-translational modifications such as proteolysis and phosphorylation, but linking modification patterns to function remains a major challenge. To obtain a global view of the biochemical state of cTn in native tissue, we performed high resolution top-down mass spectrometry of cTn heterotrimers from healthy adult rat hearts. cTn heterotrimers were affinity purified, desalted and then directly subjected to mass spectrometry using a 7 Tesla Thermo LTQ-FT-ICR instrument equipped with an ESI source. Molecular ions for N-terminally processed and acetylated cTnI and cTnT were readily detected as were other post-translationally modified forms of these proteins. cTnI was phosphorylated with a distribution of un-, mono- and bisphosphorylated forms of 41 +/- 3%, 46 +/- 1%, 13 +/- 3%, respectively. cTnT was predominantly monophosphorylated and partially proteolyzed at the Glu(29)-Pro(30) peptide bond. Also observed in high resolution spectra were 'shadow' peaks of similar intensity to 'parent' peaks exhibiting masses of cTnI+16 Da and cTnT+128 Da, subsequently shown by tandem mass spectrometry (MS/MS) to be single amino acid polymorphisms. Intact and protease-digested cTn subunits were fragmented by electron capture dissociation or collision activated dissociation to localize an Ala/Ser polymorphism at residue 7 of cTnI. Similar analysis of cTnT localized an additional Gln within a three residue alternative splice site beginning at residue 192. Besides being able to provide unique insights into the global state of post-translational modification of cTn subunits, high resolution top-down mass spectrometry readily revealed naturally occurring single amino acid sequence variants including a genetic polymorphism at residue 7 in cTnI, and an alternative splice isoform that affects a putative hinge region around residue 192 of cTnT, all of which co-exist within a single rat heart.

Phosphorylation of troponin I controls cardiac twitch dynamics: evidence from phosphorylation site mutants expressed on a troponin I-null background in mice.

The cardiac myofilament protein troponin I (cTnI) is phosphorylated by protein kinase C (PKC), a family of serine/threonine kinases activated within heart muscle by a variety of agonists. cTnI is also a substrate for cAMP-dependent protein kinase (PKA) activated during beta-adrenergic signaling. To investigate the role of cTnI phosphorylation in contractile regulation by these pathways, we generated transgenic mice harboring a mutated cTnI protein lacking phosphorylation sites for PKC (serine(43/45) and threonine(144) mutated to alanine) and for PKA (serine(23/24) mutated to alanine). Transgenic mice were interbred with cTnI-knockout mice to ensure the absence of endogenous phosphorylatable cTnI. Here, we report that regulation of myocyte twitch kinetics by beta-stimulation and by endothelin-1 was altered in myocytes containing mutant cTnI. In wild-type myocytes, the beta-agonist isoproterenol decreased twitch duration and relaxation time constant (tau) by 37% to 44%. These lusitropic effects of isoproterenol were reduced by about half in nonphosphorylatable cTnI mutant myocytes and were absent in cTnI mutants also lacking phospholamban (generated by crossing cTnI mutants with phospholamban-knockout mice). These observations are consistent with important roles for both cTnI and phospholamban phosphorylation in accelerating relaxation after beta-adrenergic stimulation. In contrast, endothelin-1 increased twitch duration by 32% and increased tau by 58%. These endothelin-1 effects were substantially blunted in nonphosphorylatable cTnI myocytes, indicating that PKC phosphorylation of cTnI slows cardiac relaxation and increases twitch duration. We propose that beta-agonists and endothelin-1 regulate cardiac twitch dynamics in opposite directions in part through phosphorylation of the myofilament protein cTnI on distinct sites.

Endothelin-1 and PKC induce positive inotropy without affecting pHi in ventricular myocytes.

It has been proposed that intracellular alkalinization underlies the enhanced contractility of ventricular myocytes exposed to endothelin (ET)-1. The effects of ET-1 on the contractility and intracellular pH (pH(i)) were examined here in cultured adult rat ventricular myocytes by employing the pH-sensitive fluorescent dye SNARF-1. Variable pH(i) changes were observed on ET-1 stimulation. Most myocytes (n = 20 of 32) did not alkalinize, but showed an approximate 60% increase in twitch amplitude in response to ET-1. In the remaining myocytes (12 of 32), ET-1 induced an increase in pH(i) by 0.05 +/- 0.02 pH units with a similar approximate 60% increase in twitch amplitude. Therefore, there was no strong correlation between ET-1-mediated positive inotropy (enhanced contractility) and intracellular alkalinization. To determine whether ET-1 contractile and pH(i) responses were mediated by protein kinase C (PKC), yellow fluorescent protein (YFP)-fused dominant negative (dn) PKC constructs were used as isoform specific inhibitors. In dn-PKC-epsilon-YFP-expressing myocytes, the ET-1-mediated positive inotropic response was greatly diminished to 13 +/- 15%, but alkalinization was still observed. Expression of dn-PKC-delta-YFP also did not block alkalinization, but in this case the positive inotropic response was still observed. In a previous study, we showed that expression of PKC-delta and PKC-epsilon caused a strong positive inotropy on stimulation with phorbol 12,13-dibutyrate (PDBu). Using this system, PDBu failed to affect pH(i) in the majority of PKC expressing myocytes despite increases in twitch amplitudes of >60%. Overall, the poor correlation of positive inotropic responses and alkalinization was observed for ET-1 with and without dn-PKC constructs and for PDBu with and without wild-type PKC constructs. These results suggest that ET-1 produces positive inotropy via PKC-epsilon by mechanisms other than intracellular alkalinization.

Endothelin-1 mobilizes profilin-1-bound PIP2 in cardiac muscle.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a key down-stream substrate of the endothelin signaling pathway and plays a role in regulating protein function at the membrane-cytoskeletal interface. However, the dynamic properties of distinct pools of PIP2 are poorly understood, especially for PIP2 that is bound to cytoskeletal proteins. We investigated the effects of endothelin-1 (ET-1) stimulation on protein-bound PIP2 in cardiac muscle. Isolated rat myocytes and homogenized mouse ventricles were exposed to 10 nM ET-1 for varying time periods and protein-bound PIP2 was analyzed using an anti-PIP2 antibody and Western blotting. Several cytoskeletal proteins were found to contain tightly bound PIP2, including profilin-1 (approximately 15 kDa), capZ (approximately 32 kDa), gCap39, (approximately 39 kDa) and alpha-actinin (approximately 106 kDa). Interestingly, ET-1 pretreatment reduced the amount of PIP2 bound to profilin-1 by 46% after 15 mins, followed by a recovery to near basal levels after 60 mins. ET-1 had no effect on capZ-, gCap39-, or alpha-actinin-bound PIP2 levels. To further explore the dynamics of PIP2 binding, brefeldin-A (BFA) was used to disrupt PIP2 binding to ADP-ribosylation factors and to impair receptor internalization. Pretreatment with 1 microM BFA increased the PIP2 signal on profilin-1 x 54% after 15 mins, followed by a decline to subbasal levels after 60 mins. Like ET-1, BFA had no effect on levels of PIP2 bound to capZ or to alpha-actinin. Taken together, the data indicate that profilin-1 binds PIP2 dynamically and may serve as a key regulator of the balance between cytoskeletal integrity and PIP2 availability for Ca2+/PKC signaling in the heart.

Endothelin receptor overexpression alters diastolic function in cultured rat ventricular myocytes.

The endothelin (ET) signaling pathway controls many physiological processes in myocardium and often becomes upregulated in heart diseases. The aim of the present study was to investigate the effects of ET receptor upregulation on the contractile function of adult ventricular myocytes. Primary cultured adult rat ventricular myocytes were used as a model system of ET receptor overexpression in the heart. Endothelin receptor type A (ETA) or type B (ETB) was overexpressed by Adenoviral infection, and the twitch responses of infected ventricular myocytes were measured after ET-1 stimulation. Overexpression of ETA exaggerated positive inotropic effect (PIE) and diastolic shortening of ET-1, and induced a new twitch response including twitch broadening. On the contrary, overexpression of ETB increased PIE of ET-1, but did not affect other two twitch responses. Control myocytes expressing endogenous receptors showed a parallel increase in twitch amplitude and systolic Ca(2+) in response to ET-1. However, intracellular Ca(2+) did not change in proportion to the changes in contractility in myocytes overexpressing ETA. Overexpression of ETA enhanced both systolic and diastolic contractility without parallel changes in Ca(2+). Differential regulation of this nature indicates that upregulation of ETA may contribute to diastolic myocardial dysfunction by selectively targeting myofi lament proteins that regulate resting cell length, twitch duration and responsiveness to prevailing Ca(2+).

A preferred AMPK phosphorylation site adjacent to the inhibitory loop of cardiac and skeletal troponin I.

5'-AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that is activated when cellular AMP to ATP ratios rise, potentially serving as a key regulator of cellular energetics. Among the known targets of AMPK are catabolic and anabolic enzymes, but little is known about the ability of this kinase to phosphorylate myofilament proteins and thereby regulating the contractile apparatus of striated muscles. Here, we demonstrate that troponin I isoforms of cardiac (cTnI) and fast skeletal (fsTnI) muscles are readily phosphorylated by AMPK. For cTnI, two highly conserved serine residues were identified as AMPK sites using a combination of high-resolution top-down electron capture dissociation mass spectrometry, (32) P-incorporation, synthetic peptides, phospho-specific antibodies, and site-directed mutagenesis. These AMPK sites in cTnI were Ser149 adjacent to the inhibitory loop and Ser22 in the cardiac-specific N-terminal extension, at the level of cTnI peptides, the intact cTnI subunit, whole cardiac troponin complexes and skinned cardiomyocytes. Phosphorylation time-course experiments revealed that Ser149 was the preferred site, because it was phosphorylated 12-16-fold faster than Ser22 in cTnI. Ser117 in fsTnI, analogous to Ser149 in cTnI, was phosphorylated with similar kinetics as cTnI Ser149. Hence, the master energy-sensing protein AMPK emerges as a possibly important regulator of cardiac and skeletal contractility via phosphorylation of a preferred site adjacent to the inhibitory loop of the thin filament protein TnI.

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.

Sustained Ca2+ signaling and delayed internalization associated with endothelin receptor heterodimers linked through a PDZ finger.

G protein-coupled receptors (GPCRs), including endothelin receptor A (ETA) and B (ETB), may form dimers or higher-order oligomers that profoundly influence signaling. Here we examined a PDZ finger motif within the C-terminus of ETA and its role in heterodimerization with ETB, and in homodimerization with itself, when expressed in HEK293 cells. Receptor dimerization was monitored by (i) fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) (FRET donor) and tetracysteine/FlAsH (FRET acceptor) fused to the C-termini of ET receptors, and (ii) coimmunoprecipitation of ET receptors after mild detergent solubilization. Mutations in a PDZ finger motif at threonine403/serine404 eliminated FRET and reduced coimmunoprecipitation of heterodimers and homodimers. Functional consequences were evaluated by measuring mobilization of intracellular Ca2+ and internalization of receptors in response to a 10 nmol/L ET-1 challenge. PDZ mutations converted a sustained Ca2+ signal mediated by ETA:ETB heterodimers into a transient response, similar to that observed for homodimers or monomers. Heterodimers containing PDZ mutations were seen to internalize in a similar time domain (approximately 5 min) to the transient Ca2+ elevation and with similar kinetics to internalization of ETA homodimers or monomers. Without the PDZ mutations, heterodimers did not internalize over 15 min, suggesting the intriguing possibility that sustained Ca2+ signaling was a consequence (at least in part) of delayed internalization. The results are consistent with structural models of ETA-receptor dimerization that place threonine403/serine404 of the PDZ finger motif at the interaction interface between heterodimers and homodimers. Sustained Ca2+ signaling and delayed endocytosis of ETA:ETB heterodimers argues strongly for a unique dimer interface that impacts transmembrane signaling and internalization.

Contractile regulation by overexpressed ETA requires intact T tubules in adult rat ventricular myocytes.

Endothelin (ET)-1 regulates the contractility and growth of the heart by binding G protein-coupled receptors of the ET type A receptor (ET(A))/ET type B (ET(B)) receptor family. ET(A), the predominant ET-1 receptor subtype in myocardium, is thought to localize preferentially within cardiac T tubules, but the consequences of mislocalization are not fully understood. Here we examined the effects of the overexpression of ET(A) in conjunction with T-tubule loss in cultured adult rat ventricular myocytes. In adult myocytes cultured for 3 to 4 days, the normally robust positive inotropic effect (PIE) of ET-1 was lost in parallel with T-tubule degeneration and a decline in ET(A) protein levels. In these T tubule-compromised myocytes, an overexpression of ET(A) using an adenoviral vector did not rescue the responsiveness to ET-1, despite the robust expression in the surface sarcolemma. The inclusion of the actin polymerization inhibitor cytochalasin D (CD) during culture prevented gross morphological changes including a loss of T tubules and a rounding of intercalated discs, but CD alone did not rescue the responsiveness to ET-1 or prevent ET(A) downregulation. The rescue of a normal PIE in 3- to 4-day cultured myocytes required both an increased expression of ET(A) and intact T tubules (preserved with CD). Therefore, the activation of ET(A) localized in T tubules was associated with a strong PIE, whereas the activation of ET(A) in surface sarcolemma was not. The results provide insight into the pathological cardiac conditions in which ET(A) is upregulated and T-tubule morphology is altered.

Interaction and inhibitory cross-talk between endothelin and ErbB receptors in the adult heart.

Endothelin-1 (ET-1) regulates contractility and growth of the mammalian heart by binding endothelin receptor type A (ET(A)) and endothelin receptor type B (ET(B)) G-protein-coupled receptors. To identify growth signaling pathways associated with ET-1 receptors in adult myocardium, a combined immunoprecipitation/proteomic analysis was performed. Signaling proteins believed to function downstream of ET(A) such as Galpha(q), phospholipase C-beta1, protein kinase C (PKC) epsilon, and PKCdelta were identified in immunoprecipitates of ET(A) by matrix-assisted laser desorption ionization/time of flight mass spectrometry. Also prominent were the growth factor receptor tyrosine kinases erbB2 and erbB4 and their downstream growth signaling effectors phosphoinositide-3 kinase (PI3 kinase), Akt, Raf-1, mitogen-activated protein kinase kinase (MEK), and extracellular signal-regulated kinase (Erk). Western blot analysis confirmed coimmunoprecipitation of erbB2/4, PI3 kinase, and Akt with ET(A), and confocal microscopy revealed their colocalization in cardiac transverse tubules (T-tubules). The erbB4 receptor ligand neuregulin-1beta (NRG1beta) promoted erbB2/4 tryosine phosphorylation and Akt serine phosphorylation in ventricular myocytes, whereas treatment with ET-1 did not. This observation argues against ET-1 growth signaling occurring via erbB2/4 transactivation in adult myocardium. ET-1 did, however, stimulate Erk1/2 phosphorylation and substantially blunted several NRG1beta-mediated actions, including erbB2/4 phosphorylation, serine phosphorylation of Akt, and negative inotropy. This inhibitory cross-talk between ET(A) and erbB2/4-Akt pathways was mimicked by a phorbol ester and blocked by pharmacological inhibition of PKC or MEK/Erk. The proteomic analysis and subsequent investigation of receptor cross-talk indicate that growth signaling between ET(A) and erbB pathways is fundamentally different in adult versus neonatal cardiac myocytes. The results may be relevant to cardiomyopathies associated with 1) prolonged exposure to ET-1; 2) degeneration of T-tubules; and 3) therapies targeted at erbB2 inhibition.