Search

The native reference peptide (NRP) method has been adapted to the measure of the degree of protein nitration at a specific tyrosine residue. In these experiments, human serum albumin was modified in a myeloperoxidase-mediated reaction in the presence of nitrite, with nitration detected predominantly at one site, [Y.sup.162]. The time-dependent increase in nitration at this site was measured based on the increasing abundance of the peptide [sup.162][Y.sup.n]LYEIA[R.sup.168] and the corresponding decrease in the [sup.162]YLYEIA[R.sup.168] peptide in in-gel trypsin digests. The peptide [sup.66]LVNEVIEFA[K.sup.75], also formed in the tryptic digest, was used as the native reference peptide. Quantitation was achieved by determining the chromatographic peak area of the two analyte peptides relative to the native reference peptide by LC/tandem mass spectrometric analyses with selected reaction monitoring. The NRP results were validated by correlation to the time-dependent increase in total protein-nitrotyrosine content determined by Western blot analysis. The precision and limit of detection of the assay were also evaluated and were found to be approximately 10% (relative standard deviation) and 5 fmol on-column, respectively. These results demonstrate the utility of the NRP method for quantitative analyses of posttranslation modifications, in terms of broad applicability, ease of experimental design, sensitivity, and precision.

We have developed and validated a method that uses liquid chromatography/electrospray ionization-mass spectrometry to quantify site-specific protein phosphorylation. The method uses selected ion monitoring to determine the chromatographic peak areas of specific tryptic peptides from the protein of interest. The extent of phosphorylation is determined from the ratio of the phosphopeptide peak area to the peak area of an unmodified reference peptide that acts as internal standard, correcting for variations in protein amounts and peptide recovery in the digest preparation procedure. As a result, we refer to this protocol as the native reference peptide method. Mole of phosphate at the selected site per mole of protein is obtained from this ratio, using calibration curves of synthetic peptides to determine relative responses. Our method begins with protein separation by SDS-PAGE and is carried out on amounts of peptide produced by an ingel digestion of single Coomassie blue-stained bands. To illustrate the utility of the method and provide validation, we used cardiac troponin I as analyte and monitored the time course of a protein kinase C [beta]II reaction. Those analyses appropriately demonstrate the time-dependent increase of phosphorylation at a PKC-preferred site, Ser44 in the peptide [sup.41][ISAS.sup.p][R.sup.45] and the concomitant consumption of the nonphosphorylated peptide. We believe that this method provides a novel tool to directly measure specific phosphorylation sites in proteins in different physiological states and expect that the method will be adaptable not only to a variety of samples types (i.e., culture cells, tissues, etc.) but to a variety of posttranslation modifications as well.

The COOH terminus of troponin I is essential in the binding of troponin C, T, and I in the presence of calcium. The COOH terminus is exposed in the muscle thin filament and participates in the allosteric switch during muscle contraction.

Spontaneously hypertensive rats (SHR) are used to evaluate the myofilament calcium sensitivity-associated alteration in troponin-1 (Tn-1) phosphorylation and the expression of troponin isoforms during the evolution to decompensated cardiac hypertrophy. Results indicate that Tn-1 phosphorylation upon beta adrenergic stimulation of myocytes decreased among SHRs. The implications of these findings are discussed.

A study was performed to compare the baseline and protein kinase A (PKA)-dependent troponin I (TnI) phosphorylation in left ventricular myocytes from hearts of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Phosphorylation of TnI by PKA was believed to reduce myofilament Ca2+ sensitivity. Results indicated that after activation of the beta-adrenergic mechanism, PKA-dependent TnI phosphorylation from the SHR group was greater than in WKY coupled with decreased Ca2+ sensitivity of actomyosin ATPase activity.

A study was initiated to determine effects of mitochondrial metabolism on mitochondrial calcium (Ca) uptake during physiological processes. Mensuration of changes in the left ventricular function, pyruvate dehydrogenase (PDH) activity and mitochondrial Ca content was done in perfused hamster hearts frozen during systole. Results indicated that no relevant change was seen in mitchondrial Ca content during isoproterenol activation and increased perfusion pressure.

Diseases of striated muscle linked to intermediate filament (IF) proteins are associated with defects in the organization of the contractile apparatus and its links to costameres, which connect the sarcomeres to the cell membrane. Here we study the role in skeletal muscle of synemin, a type IV IF protein, by examining mice null for synemin (synm-null). Synm-null mice have a mild skeletal muscle phenotype. Tibialis anterior (TA) muscles show a significant decrease in mean fiber diameter, a decrease in twitch and tetanic force, and an increase in susceptibility to injury caused by lengthening contractions. Organization of proteins associated with the contractile apparatus and costameres is not significantly altered in the synm-null. Elastimetry of the sarcolemma and associated contractile apparatus in extensor digitorum longus myofibers reveals a reduction in tension consistent with an increase in sarcolemmal deformability. Although fatigue after repeated isometric contractions is more marked in TA muscles of synm-null mice, the ability of the mice to run uphill on a treadmill is similar to controls. Our results suggest that synemin contributes to linkage between costameres and the contractile apparatus and that the absence of synemin results in decreased fiber size and increased sarcolemmal deformability and susceptibility to injury. Thus synemin plays a moderate but distinct role in fast twitch skeletal muscle. [ABSTRACT FROM AUTHOR]

A-kinase anchoring proteins (AKAPs) bind the regulatory subunits of protein kinase A (PKA) and localize the holoenzyme to discrete signaling microdomains in multiple subcellular compartments. Despite emerging evidence for a nuclear pool of PKA that rapidly responds to activation of the PKA signaling cascade, only a few AKAPs have been identified that localize to the nucleus. Here we show a PKA-binding domain in the amino terminus of Chd8, and demonstrate subcellular colocalization of Chd8 with RII. RII overlay and immunoprecipitation assays demonstrate binding between Chd8-S and RIIa. Binding is abrogated upon dephosphorylation of RIIα. By immunofluorescence, we identified nuclear and perinuclear pools of Chd8 in HeLa cells and rat neonatal cardiomyocytes. We also show high levels of Chd8 mRNA in RNA extracted from post- natal rat hearts. These data add Chd8 to the short list of known nuclear AKAPs, and implicate a function for Chd8 in post-natal rat cardiac development. [ABSTRACT FROM AUTHOR]

Intermediate filaments (IFs) in cardiomyocytes consist primarily of desmin, surround myofibrils at Z disks, and transmit forces from the contracting myofilaments to the cell surface through costameres at the sarcolemma and desmosomes at intercalated disks. Synemin is a type IV IF protein that forms filaments with desmin and also binds α-actinin and vinculin. Here we examine the roles and expression of the α and β forms of synemin in developing rat cardiomyocytes. Quantitative PCR showed low levels of expression for both synemin mRNAs, which peaked at postnatal day 7. Synemin was concentrated at sites of cell--cell adhesion and at Z disks in neonatal cardiomyocytes. Overexpression of the individual isoforms showed that α-synemin preferentially localized to cell-cell junctions, whereas β-synemin was primarily at the level of Z disks. An siRNA targeted to both synemin isoforms reduced protein expression in cardiomyocytes by 70% and resulted in a failure of desmin to align with Z disks and disrupted cell--cell junctions, with no effect on sarcomeric organization. Solubility assays showed that α-synemin was soluble and interacted with sarcomeric-actinin by coimmunoprecipitation, while α-synemin and desmin were insoluble. We conclude that β-synemin mediates the association of desmin IFs with Z disks, whereas-synemin stabilizes junctional complexes between cardiomyocytes. [ABSTRACT FROM AUTHOR]

Identifies a gene expression fingerprint for heart failure. Statistical method to establish comprehensive cutoff point for identification of differentially-expressed genes; Differentiation of etiologies of heart failure by gene expression fingerprint; Gene clusters that show coordinated up- or downregulation in human heart failure.

Synemin binds PKA RII via blot overlays and co-immunoprecipitates with RII from adult rat cardiomyocytes (CM) through an amphipathic helical AKAP domain. We also show colocalization of synemin with RII at Z-lines and the perinuclear region using immunohistochemistry with adult CM. In isolated rat neonatal CMs, synemin localized to the sarcolemma but not to filamentous, cytoplasmic desmin. There are two known isoforms of synemin in rat (H and M) that differ by 932bp in the C-terminus. Using antibodies to each isoform, the two differ in their subcellular location: H is at the sarcolemma, while M is at striations. A polyclonal antibody against H recognizes 210kDa and 100kDa bands (SDS-PAGE weights) via western blots of CM lysates, suggesting a new putative isoform. Studies by other labs demonstrated an L isoform comprising the rod domain (exons 1-3) and a truncated tail (exon 5) in mouse brain, skeletal muscle, and heart that may be expressed in rat neonatal or adult CMs. Using affinity-tagged constructs, we are isolating the multimolecular protein complexes formed by each isoform. We hypothesize that these isoforms have variable subcellular locations and binding partners and may be temporally regulated. We conclude that synemin isoforms have different functions and target PKA near different substrates within myocytes. [ABSTRACT FROM AUTHOR]

Abstract: Background: The underlying molecular mechanisms of the remodeling after myocardial infarction (MI) remain unclear. The purpose of this study was to investigate the role of a survival pathway (PI3K/Akt) and an apoptosis pathway (calcineurin/BAD) in the remodeling after MI in a large animal model. Methods: Ten Dorset hybrid sheep underwent 25% MI in the left ventricle (LV, n=10). Five sheep were used as sham control. The regional strain was calculated from sonomicrometry. Apoptosis and the activation of the PI3K/Akt and calcineurin/BAD pathways were evaluated in the non-ischemic adjacent zone and the remote zone relative to infarct by immunoblotting, immunoprecipitation, and immunofluorescence staining. Results: Dilation and dysfunction of LV were present at 12weeks after MI. The regional strain in the adjacent zone was significantly higher than in the remote zone at 12weeks (36.6±4.0% vs 9.5±3.6%, p< 0.05). Apoptosis was more severe in the adjacent zone than in the remote zone. The PI3K/Akt and calcineurin/BAD pathways were activated in the adjacent zone. Dephosphorylation and translocation of BAD were evident in the adjacent zone. Regional correlation between the strain and the expression of calcineurin/BAD indicated that the activation was strain-related (R 2 =0.46, 0.48, 0.39 for calcineurin, BAD, mitochondrial BAD, respectively, p <0.05). Conclusions: The PI3K/Akt survival and calcineurin/BAD apoptotic pathways were concomitantly activated in the non-ischemic adjacent zone after MI. The calcineurin/BAD pathway is strain related and its imbalanced activation may be one of the causes of progressive remodeling after MI. [Copyright &y& Elsevier]

Protein kinase A (PKA)-dependent phosphorylation is regulated by targeting of PKA to its substrate as a result of binding of regulatory subunit, R, to A-kinase-anchoring proteins (AKAPs). We investigated the effects of disrupting PKA targeting to AKAPs in the heart by expressing the 24-amino acid regulatory subunit RII-binding peptide, Ht31, its inactive analog, Ht31P, or enhanced green fluorescent protein by adenoviral gene transfer into rat hearts in vivo. Ht31 expression resulted in loss of the striated staining pattern of type II PKA (RII), indicating loss of PKA from binding sites on endogenous AKAPs. In the absence of isoproterenol stimulation, Ht31-expressing hearts had decreased +dP/dt[submax] and -dP/dt[submin] but no change in left ventricular ejection fraction or stroke volume and decreased end diastolic pressure versus controls. This suggests that cardiac output is unchanged despite decreased +dP/dt and -dP/dt. There was also no difference in PKA phosphorylation of cardiac troponin I (cTnI), phospholamban, or ryanodine receptor (RyR[sub2]). Upon isoproterenol infusion, +dP/dt[submax] and -dP/dt[submin] did not differ between Ht31 hearts and controls. At higher doses of isoproterenol, left ventricular ejection fraction and stroke volume increased versus isoproterenol-stimulated controls. This occurred in the context of decreased PKA phosphorylation of cTnI, RyR[sub2], and phospholamban versus controls. We previously showed that expression of N-terminal-cleaved cTnI (cTnIND) in transgenic mice improves cardiac function. Increased cTnI N-terminal truncation was also observed in Ht31-expressing hearts versus controls. Increased cTnI-ND may help compensate for reduced PKA phosphorylation as occurs in heart failure. [ABSTRACT FROM AUTHOR]

Subcellular compartmentalization of the cAMP-dependent protein kinase (PKA) by protein kinase A-anchoring proteins (AKAPs) facilitates local protein phosphorylation. However, little is known about how PKA targeting to AKAPs is regulated in the intact cell. PKA binds to an amphipathic helical region of AKAPs via an N-terminal domain of the regulatory subunit. In vitro studies showed that autophosphorylation of type II regulatory subunit (RII) can alter its affinity for AKAPs and the catalytic subunit (PKAcat). We now investigate whether phosphorylation of serine 96 on RII regulates PKA targeting to AKAPs, downstream substrate phosphorylation and calcium cycling in primary cultured cardiomyocytes. We demonstrated that, whereas there is basal phosphorylation of RII subunits, persistent maximal activation of PKA results in a phosphatase-dependent loss of RII phosphorylation. To investigate the functional effects of RI! phosphorylation, we constructed adenoviral vectors incorporating mutants which mimic phosphorylated (RIIS96D), nonphosphorylated (RIIS96A) RII or wild-type (WT) RII and performed adenoviral infection of neonatal rat cardiomyocytes. Coimmunoprecipitation showed that more AKAP15/18 was pulled down by the phosphomimic, RIIS96D, than RIIS96A. Phosphorylation of phospholamban and ryanodine receptor was significantly increased in cells expressing RIIS96D versus RIIS96A. Expression of recombinant RII constructs showed significant effects on cytosolic calcium transients. We propose a model illustrating a central role of RII phosphorylation in the regulation of local PKA activity. We conclude that RII phosphorylation regulates PKA-dependent substrate phosphorylation and may have significant implications for modulation of cardiac function. [ABSTRACT FROM AUTHOR]

Abstract: The need to assess heart failure at an early stage highlights the importance of accurate microarray analysis using small tissue samples. To test our ability to obtain high quality RNA from biopsy-sized cardiac specimens, amplification was performed on RNA from biopsy-sized samples of left ventricle (LV) tissue from one explanted failing human heart and one non-failing heart. Two methods were used: one-cycle (1C) amplification of 1.6 μg of RNA, and two-cycle (2C) amplification of 50 ng of RNA. The resulting cRNA was hybridized to Affymetrix GeneChip® arrays. Over 65% of all differentially expressed genes for failing vs non-failing hearts were concordant between 1C and 2C RNA amplification. Differentially expressed genes between 1C and 2C RNA amplification in our study were highly correlated (R 2 = 0.957 and changes in gene expression agreed with prior studies on genes and heart failure; e.g., decreased α-myosin heavy chain and α-tropomyosin, as well as increased expression of insulin-like growth factor). Two cycles of amplification from cardiac biopsies will permit accurate transcription profiling of heart failure at pre-symptomatic stages. Ability to measure gene expression from nanogram amounts of RNA will provide new opportunities to predict progression to symptomatic heart failure, and to identify potential targets for therapy. [Copyright &y& Elsevier]

Abstract: Targeting of protein kinase A (PKA) by A-kinase anchoring proteins (AKAPs) contributes to high specificity of PKA signaling pathways. PKA phosphorylation of myofilament and cytoskeletal proteins may regulate myofibrillogenesis and myocyte remodeling during heart disease; however, known cardiac AKAPs do not localize to these regions. To identify novel AKAPs which target PKA to the cytoskeleton or myofilaments, a human heart cDNA library was screened and the intermediate filament (IF) protein, synemin, was identified as a putative RII (PKA regulatory subunit type II) binding protein. A predicted RII binding region was mutated and resulted in loss of RII binding. Furthermore, synemin co-localized with RII in SW13/cl.1-vim+ cells and co-immunoprecipitated with RII from adult rat cardiomyocytes. Synemin was localized at the level of Z-lines with RII and desmin in adult hearts, however, neonatal cardiomyocytes showed differential synemin and desmin localization. Quantitative Western blots also showed significantly more synemin was present in failing human hearts. We propose that synemin provides temporal and spatial targeting of PKA in adult and neonatal cardiac myocytes. [Copyright &y& Elsevier]

Besides the core structure conserved in all troponin I isoforms, cardiac troponin I (cTnI) has an N-terminal extension that contains phosphorylation sites for protein kinase A under β-adrenergic regulation. A restricted cleavage of this N-terminal regulatory domain occurs in normal cardiac muscle and is up-regulated during hemodynamic adaptation (Z.-B. Yu, L.-F. Thang, and J.-P. Jin (2001) J. Biol Chem. 276, 15753–15760). In the present study, we developed transgenic mice over-expressing the N-terminal truncated cTnI (cTnI-ND) in the heart to examine its biochemical and physiological significance. Ca2+-activated actomyosin ATPase activity showed that cTnI-ND myofibrils had lower affinity for Ca2+ than controls, similar to the effect of isoproterenol treatment. In vivo and isolated working heart experiments revealed that cTnl-ND hearts had a significantly faster rate of relaxation and lower left ventricular end diastolic pressure compared with controls. The higher baseline relaxation rate of cTnI-ND hearts was at a level similar to that of wild type mouse hearts under β-adrenergic stimulation. The decrease in cardiac output due to lowered preload was significantly smaller for cTnI-ND hearts compared with controls. These findings indicate that removal of the N-terminal extension of cTnI via restricted proteolysis enhances cardiac function by increasing the rate of myocardial relaxation and lowering left ventricular end diastolic pressure to facilitate ventricular filling, thus resulting in better utilization of the Frank-Starling mechanism. [ABSTRACT FROM AUTHOR]

Demonstrates that the targeting of protein kinase A by muscle A kinase-anchoring protein (mAKAP) regulates phosphorylation. Effect of mAKAP in the function of the skeletal muscle ryanodine receptor; Co-distribution of ryanodine receptor1 and mAKAP at the perinuclear space; Role of mAKAP expression in increasing protein kinase A-dependent calcium release by ryanodine receptor1 from internal stores.