Phosphoproteome Analysis of Cardiomyocytes Subjected to β-Adrenergic Stimulation
Identification and Characterization of a Cardiac Heat Shock Protein p20
Posttranslational modification of target substrates underlies biological processes through activation/inactivation of signaling cascades. To concurrently identify the phosphoprotein substrates associated with cardiac β-adrenergic signaling, the mouse myocyte phosphoproteome was analyzed using 2-D gel electrophoresis in combination with 32P autoradiography. Phosphoprotein spots, detected by silver staining, were identified using MALDI-TOF mass spectrometry in conjunction with computer-assisted protein spot matching. Stimulation with isoproterenol (1 μmol/L for 5 minutes) was associated with maximal increases in myocyte contractile parameters, and significant stimulation of the phosphorylation of troponin I (190±23%) and succinyl CoA synthetase (160±16%), whereas the phosphorylation of pyruvate dehydrogenase (48±10%), NADH-ubiquinone oxidoreductase (46±6%), heat shock protein 27 (18±3%), αB-crystallin (20±3%), and an unidentified 26-kDa protein (29±7%) was significantly decreased, compared with unstimulated cells (100%). After sustained (30 minutes) stimulation with isoproterenol, only the alterations in the phosphorylation levels of troponin I and NADH-ubiquinone oxidoreductase were maintained and de novo phosphorylation of a phosphoprotein (≈20 kDa and pI 5.5) was observed. The tryptic peptide fragments of this phosphoprotein were sequenced using postsource decay mass spectrometry, and the protein was subsequently cloned and designated as p20, based on its high sequence homology with rat and human skeletal p20. The mouse cardiac p20 contains the conserved domain sequences for heat shock proteins, and the RRAS consensus sequence for cAMP-PKA substrates. LC-MS/MS phosphorylation mapping confirmed phosphorylation of Ser16 in p20 on β-agonist stimulation. Adenoviral gene transfer of p20 was associated with significant increases in contractility and Ca transient peak in adult rat cardiomyocytes, suggesting an important role of p20 in cardiac function. These findings suggest that cardiomyocytes undergo significant posttranslational modification via phosphorylation in a multitude of proteins to dynamically fine-tune cardiac responses to β-adrenergic signaling.
β-Adrenergic receptors and their signaling effectors are key mediators of neurohormonal influence over the cardiovascular system, modulating the strength, velocity, and frequency of cardiac contraction and relaxation.1 At the subcellular level, the cardiac responses to β-adrenergic agonists are associated with phosphorylation of several downstream target phosphoproteins. Multiple studies have indicated that the most critical substrates for the positive inotropic and lusitropic β-agonist effects are phospholamban in the sarcoplasmic reticulum (SR) and troponin I (TnI) in the myofilaments, both phosphorylated through the cAMP-dependent protein kinase A (PKA) pathway.2–4 Phosphorylation of phospholamban relieves its inhibitory effects on SERCA2, with subsequent acceleration of SR Ca2+ transport, and phosphorylation of TnI reduces the myofilament sensitivity to Ca2+, synergistically contributing to increased relaxation rates. Other proteins involved in cardiac excitation-contraction coupling, including C-protein, the L-type Ca2+ channel, and the ryanodine receptor, are also subjected to β-agonist–induced phosphorylation.5
Sustained activation of the β-adrenergic signaling pathway can exert deleterious effects on the myocardium, promoting hypertrophy, left ventricular dysfunction,6 apoptosis,7 and heart failure.1,5 In animal models, transgenic overexpression of β1-adrenergic receptor was associated with impaired cardiac function and ventricular fibrosis.6,8 Chronic stimulation of β1-AR has also been reported to contribute to disease progression and mortality in both animal models and human patients of heart failure.1,5 Furthermore, recent evidence indicates that overstimulation of the β1-AR pathway in cardiomyocytes may be proapoptotic.7 Although a variety of players involved in β-adrenergic signaling, including protein kinases and phosphatases, have been identified, the intracellular target substrates and signaling molecules underlying β-adrenergic–mediated cardiac responses and remodeling remain elusive.
With ever-increasing knowledge of the complexity of the β-adrenergic signaling network, it is now apparent that β-agonist stimulation results in numerous alterations in signaling and regulatory proteins in the myocardium. Identifying these proteins and understanding their functional relationships will illuminate the mechanisms underlying cardiac function and dysfunction. Because phosphorylation is one of the most important posttranslational modifications for signal transduction and regulation of biological function, we applied proteomic techniques in combination with phospholabeling to analyze β-agonist–evoked protein phosphorylation profiles in mouse cardiomyocytes. We report in this study distinct phosphorylation profiles of myocardial proteins in response to acute versus sustained β-adrenergic stimulation, and the identification and characterization of a cardiac isoform of p20 associated with β-adrenergic signaling in adult rodent cardiomyocytes.
Materials and Methods
Mouse left ventricular myocytes were isolated,9 32P-labeled,3 and proteins were extracted for 2-dimensional (2-D) gel electrophoresis. 2-D gel images were analyzed using the ImageMaster 2D Elite software. The protein spots of interest were excised and their tryptic peptides were subjected to MALDI-TOF or LC-MS/MS for identification. Animals used in this study were 3- to 4-month-old FVB/N male mice (Charles River Laboratories, Wilmington, Mass) and were handled and maintained according to protocols by the ethics committee of the University of Cincinnati. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the NIH.
For a detailed description of all the methods used, please refer to the expanded Materials and Methods section available in the online data supplement at http://www.circresaha.org.
Detection and Identification of Phosphoproteins Using 2-D Gel Electrophoresis and Mass Spectrometry
Isolated left ventricular myocytes were 32P-labeled and the proteins were solubilized and separated by 2-D gel electrophoresis. Immobilized pH gradient (pH 3 to 10) strips were used for the first-dimensional isoelectric focusing (IEF), and the horizontal second-dimensional SDS-PAGE was performed following IEF. 32P autoradiography of 2-D gels revealed the presence of ≈120 phosphorylated protein spots (Figure 1A). When the same gel was subsequently silver-stained, ≈500 protein spots were visualized (Figure 1B). Several phosphorylation signal spots, detected by 32P autoradiography, were not observed by silver staining, even when protein loading was increased to 1 mg, reflecting the higher sensitivity of 32P autoradiography versus silver staining for phosphoprotein detection. Computer-assisted spot matching and overlay of the 32P autoradiograms and silver staining images of the same gel, enabled localization of phosphoprotein spots. Phosphoproteins detected by silver staining were excised and subjected to tryptic peptide mass fingerprinting (MALDI-TOF) for identification. As shown in the Table, nine phosphoproteins were positively identified, indicating that these proteins undergo dynamic posttranslational modification via phosphorylation in cardiomyocytes under basal conditions. Furthermore, identification of myosin light chain-2 (MLC-2), αB-crystallin, TnI, heat shock protein 27, and TnT was confirmed by 2-D Western blotting in conjunction with their specific antibodies (data not shown).
Phosphorylation Profiles Evoked by β-Adrenergic Activation
β-Agonist stimulation was associated with significant increases in myocyte cell shortening fraction and the rates of myocyte contraction and relaxation (Figures 2A and 2B). The time course of the myocyte contractile responses to ISO stimulation indicated that the myocyte contractile parameters reached maximal values within 5 minutes after application of ISO. The fractional shortening and rates of shortening and relengthening were increased by 1.8, 2.2, and 1.9-fold, respectively, compared with controls. The increases in the myocyte mechanical parameters were sustained for at least 30 minutes of ISO stimulation (Figures 2A and 2B). These data confirmed that on β-adrenergic stimulation, cardiomyocyte contractility was significantly enhanced, and the hypercontractile state was maintained up to 30 minutes under the conditions used.
To identify phosphoproteins involved in the β-adrenergic responses, isolated left ventricular myocytes were labeled with 32P in the absence (Figure 1A) or presence (Figure 1C) of ISO (1 μmol/L for 5 or 30 minutes), processed for 2-D gel electrophoresis and subjected to 32P autoradiography. A pairwise analysis of gels was performed and greater than 99% of the detected spots were matched, illustrating the reproducibility of 2-D electrophoresis. The normalized intensity values for each matched gel spot pair were compared with quantitate relative phosphorylation signal levels. ISO stimulation (Figure 1C) was associated with significant alterations in 32P incorporation compared with nonstimulated (basal) cardiomyocytes (Figure 1A). However, analysis of the silver-stained images (≈500 protein spots) revealed no significant differences in respective signal intensities between ISO-treated (Figure 1D) and control (Figure 1B) myocytes. The phosphoprotein spots with significant alterations in their 32P signal abundance on ISO were subsequently identified. Phosphorylation of TnI was doubled on brief (5 minutes) stimulation by ISO, and remained high after 30 minutes of ISO stimulation (Figures 3 and 4⇓A). There was a basal level of phosphorylation of TnT, α-tropomyosin (α-TM), and MLC-2, which was not altered on ISO stimulation (Figures 3 and 4⇓A). Interestingly, image analysis indicated that the phosphoprotein levels of several mitochondrial enzymes were altered (Figures 3 and 4⇓B). Phosphorylation of pyruvate dehydrogenase (PDH) was decreased by 50%, whereas phosphorylation of succinyl CoA synthetase (SCS) was increased significantly (Figures 1C and 4⇓B) in response to 5 minutes of ISO. After 30 minutes of ISO stimulation, the phosphorylation levels of both PDH and SCS were restored to baseline values (Figures 3 and 4⇓B). Phosphorylation levels of complex I were dramatically decreased in response to 5 minutes of ISO stimulation (Figure 1C) and remained stable for at least 30 minutes (Figures 3 and 4⇓B). Furthermore, phosphorylation of the chaperone proteins HSP27 and αB-crystallin was mildly, but significantly, decreased on brief β-adrenergic stimulation (Figures 1C and 4⇓C), whereas prolonged ISO treatment restored the phosphoprotein levels to control values in unstimulated cardiomyocytes (Figures 3 and 4⇓C). Similarly, the phosphorylation level of a protein with MW 26 kDa and pI 8.0 (UPP) was significantly downregulated by brief but not prolonged ISO stimulation (Figures 1C, 3B, and 4⇓⇓C). Identification of this protein by mass spectrometry was unsuccessful due to its extremely low abundance. In addition, to confirm the changes in the degree of protein phosphorylation, obtained from 2-D 32P autoradiography, we selected TnI as an example and assessed its phosphorylation levels by a different method, ie, normalizing its Pi signal (autoradiography) to its protein signal (Western blotting) (see online data supplement).
Phosphorylation of phospholamban has been postulated to be a key mediator of the positive inotropic and lusitropic actions of β-adrenergic stimulation.3,5,9 However, phospholamban was not detected by 32P autoradiography or silver staining of the 2-D gels, possibly due to its low abundance and/or high hydrophobicity. Thus, quantitative immunoblotting was performed, using phospholamban phosphorylation site-specific antibodies, to confirm the phosphorylation status of phospholamban in the myocytes (Figures 4D and 4E). On application of ISO for 5 minutes, the phosphorylation levels of Ser16 and Thr17 in phospholamban were significantly increased. Interestingly, the relative increase in Ser16 phosphorylation (≈16-fold) was much higher than that in Thr17 phosphorylation (≈5-fold). After 30 minutes of β-adrenergic stimulation, Ser16 phosphorylation was significantly decreased, whereas Thr17 phosphorylation was not significantly diminished. However, the phosphorylation levels at both Ser16 and Thr17 sites remained significantly higher than control values (Figure 4E).
De Novo Phosphorylation After Prolonged β-Adrenergic Stimulation
32P autoradiography in combination with the proteomic approach provided a powerful platform to identify phosphoproteins or de novo phosphorylations. Indeed, 30 minutes of ISO stimulation revealed a distinct phosphoprotein signal with an apparent MW of ≈20 kDa and pI of 5.5 (Figure 3B) in cardiac myocytes. This spot was consistently detected and was the only de novo phosphorylation signal observed under the conditions used. Interestingly, this phosphorylation signal was undetectable in either unstimulated cardiomyocytes (Figure 1A) or after 5 minutes of ISO stimulation (Figure 1C). To confirm and clearly resolve this phosphoprotein, 2-D zoom gel was performed using IEF strips with a narrower pH range (pH 4 to 7) (Figure 5). This subproteome approach allowed enrichment of the de novo phosphoprotein signal (Figure 5C). A distinct silver-stained protein spot colocalized with the phosphorylation signal from 32P autoradiography in myocytes-treated with ISO for 30 minutes (Figure 5D) but not in the nonstimulated (Figure 5B) or 5 minutes ISO-stimulated (Figure 1C) cells. In addition, the phosphorylation of this protein appeared to be specific to the β-adrenergic signaling pathway, because exposure of myocytes to 0.1 μmol/L 4-β phorbol 12-myristate 13-acetate (PMA) for 30 minutes, which activates the PKC isozymes, failed to cause increased phosphorylation (data not shown).
To identify the de novo phosphoprotein, the corresponding protein spot was excised from the silver-stained gel and subjected to trypsin digestion. Mass spectra of tryptic peptides from the digests of the ISO-induced de novo phosphoprotein did not yield enough peaks to assign protein identification with high confidence. However, two peaks (MH+=678.35 and MH+=1592.7) were of significant intensity to produce sequence fragment spectra via postsource decay (PSD) (Figures 5E and 5F). The MS-Tag module of the Protein Prospector (http://prospector.ucsf.edu) was utilized in extracting sequence information from the fragment ion spectra. Database search indicated that the sequences had a significant similarity with the rat skeletal heat shock protein-like p20.
Cloning and Bioinformatic Analysis of a Cardiac Isoform of p20
Based on the sequence information obtained from the mass spectrometric analysis, in combination with previously published sequences of rat skeletal p20 (GenBank accession no. D29960) and a mouse EST sequence (GenBank accession no. BF016741), two primers (forward primer, 5′-CCAGGTTTCTCTGCTCCGGGACGC-3′; and reverse primer, 5′-CGGCGGTGGAACTCTCGAGCAATG-3′) were designed and used to screen a mouse cardiac cDNA library (Incyte Genomics) by PCR methodology. Three positive cDNA clones (approximately 1.5 kb) were identified, and subsequently sequenced. Sequence analysis indicated that all of the three clones contained the full-length cDNA sequence with identical coding regions (Figure 6A) and its deduced amino acids corresponded to a MW of 17.4 kDa, with a pI of 5.45. Comparison of its derived amino acid sequence with rat and human skeletal p20 revealed 95% and 87% homology, respectively (Figure 6B). This cardiac phosphoprotein is therefore potentially a mouse cardiac analogue of p20. Interestingly, all have internal sequences highly homologous to those of the molecular chaperones small heat shock proteins (eg, HSP27) and αB-crystallin. An additional search of the Conserved Domain Database (http://www.ncbi.nlm.gov/structure/cdd) confirmed the presence of a heat shock protein domain in mouse cardiac p20 (Figure 6B), which matched 82% with the consensus sequence (106 AA, CD no. pfam00011) of heat shock proteins. In addition, the consensus sequence RRAS, predicted for cAMP-dependent phosphorylation substrates (PROSITE accession no. PS00004), was present in the mouse cardiac p20 and conserved in rat and human skeletal p20 (Figure 6B), suggesting that Ser16 in p20 is phosphorylated by cAMP-PKA during β-adrenergic stimulation in cardiomyocytes.
Identification and Phosphorylation Mapping of the Cardiac p20 via LC-MS/MS
To further confirm identification of p20 and its phosphorylation on ISO stimulation, we performed 2-D zoom gels (IEF strips, 18 cm and pH 5 to 6) to improve protein separation and minimize potential contamination. One milligram of the ISO-treated and control protein samples were run in parallel. The cardiac p20 was detected only in the ISO-treated cells by Sypro-Ruby staining (Figure 6C). However, when the 2-D gels were probed with a p20 antibody, one immunoreactive p20 spot was detected in the control myocytes. Furthermore, one immunoreactive p20 spot was also observed in the ISO-treated myocytes, with a significant pI shift to the acid side compared with controls (Figure 6D). These findings indicate the following: (1) the protein expression level of p20 is extremely low in control samples, below the detection limit of Sypro-Ruby staining, whereas immunoblotting is sensitive enough to pick up the signal; (2) the significant shift in pI of p20 suggests phosphorylation of p20 in response to ISO stimulation; and (3) p20 appears fully dephosphorylated in controls, but fully phosphorylated on maximal ISO stimulation (1 μmol/L for 30 minutes). To further verify that p20 was actually phosphorylated by ISO, the candidate “phosphorylated” p20 protein spot, detected by 2-D zoom gels in ISO-treated myocytes (Figure 6C), was excised and subjected to high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) and SALSA (scoring algorithm for spectral analysis) for identification and phosphorylation mapping. Tandem MS spectra of peptides were analyzed with Sequest, a program that allows the correlation of experimental tandem MS data with theoretical spectra generated from known protein sequences. The criteria used for positive peptide identification are the same as previously described.10 All matched peptides (online Table 2 and online Figure 2, available at http://www.circresaha.org), confirmed by visual examination of the spectra, indicated identification of p20. In addition, the spectra from potentially phosphorylated protein spots were subjected to analysis using SALSA, a pattern recognition program that can detect specific features in tandem MS spectra.11 SALSA was used to interrogate for the loss of 98 amu [ie, loss of phosphoric acid (H3PO4) from serine or threonine residue] from precursor peptide ions undergoing fragmentation in the mass spectrometer. The tandem mass spectrum of a phosphopeptide from p20 phosphorylated at Ser16, along with the expected b and y fragment ions for that peptide, are shown in Figure 6E. Collectively, the LC-MS/MS and SALSA analysis positively identified this candidate protein as p20 and residue Ser16 as the phosphorylation site.
Effect of Cardiac p20 on Cardiomyocyte Contractility and Ca2+ Transients
To determine whether the mouse cardiac p20 may affect cardiac contractility, a recombinant adenovirus encoding wild-type p20 was constructed, transduced into adult rat ventricular myocytes and cell shortening as well as intracellular Ca2+ transients were assessed. The resting cell length in p20-infected myocytes was not significantly altered, compared with the β-gal control myocytes. However, the amplitude of basal cell contraction was significantly increased in p20 infected myocytes, compared with β-gal controls (Figure 7), whereas the times to 50% and 90% relaxation were not altered. Similarly, Ca2+ transient measurements also revealed that the adenoviral gene transfer of p20 significantly increased the Ca2+ transient amplitude (Figure 7) without altering the time to 50% decay of Ca2+ transients.
Posttranslational modification via phosphorylation underlies various biological processes and particularly the propagation of cellular signals through differential activation or inactivation of specific signaling cascades. This study presents the first phosphoproteome analysis of mouse cardiomyocytes and its changes in the context of β-adrenergic stimulation. Our findings indicate the following: (1) a plethora of protein substrates undergo posttranslational modification via phosphorylation in unstimulated cardiomyocytes, including myofibrillar, mitochondrial, and chaperone proteins; (2) acute and prolonged stimulation by β-agonists differentially regulate the phosphorylation status of key substrate proteins in the various subcellular compartments, contributing to their stimulatory responses; (3) prolonged stimulation of the β-adrenergic signaling pathway induces de novo phosphorylation of a cardiac heat shock protein p20; and (4) adenovirus-mediated overexpression of p20 in adult rat myocytes is associated with increased cell contraction and Ca2+ transients. This approach for analysis of the cardiac phosphoproteome and its dynamic regulation by β-adrenergic signaling provides unique insights into the molecular mechanisms underlying modulation of cardiac function.
Phosphorylation of contractile proteins, specifically TnI, which decreases the Ca2+ affinity of troponin C, has been shown to play a key role in the relaxant effects of β-agonists in the heart.3,5 Previous studies in isolated mouse cardiomyocytes showed that phosphorylation of TnI reached a maximum (≈2-fold) after 2 minutes of ISO (1 μmol/L) stimulation, and this change correlated well with the increases in contractile parameters.3 Consistent with these observations, phosphorylation of TnI was also increased by 2-fold on brief ISO stimulation, and it remained high for at least 30 minutes of stimulation in mouse myocytes. In spite of significant alterations in the phosphorylation status of TnI, the phosphorylation levels of other major myofibrillar phosphoproteins, including MLC-1, MLC-2, TnT, and α-tropomyosin, were not altered on brief or prolonged ISO stimulation.
Subcellular compartment-specific protein phosphorylation has been proposed as a key mechanism for cells to transmit extracellular signals to cellular responses by various PKA pathways. The present investigation presents strong evidence that the mitochondrion is one of the primary targets, where PKA may engage in signal transduction. ISO stimulation of cardiac myocytes resulted in significant alterations in the phosphorylation levels of several mitochondrial enzymes including PDH, SCS, and complex I. In the ISO-treated myocytes, the levels of phosphorylated (inactive) PDH were decreased by ≈50%, indicating increases in both the active fraction of the enzyme and the rate of ATP synthesis via oxidative phosphorylation. These findings are consistent with a previous report on ISO stimulation of Langendorff-perfused hamster hearts,12 which was associated with ≈2-fold increase in PDH activity. By contrast, in genetically diabetic mice, increases in phosphorylation of PDH correlated well with decreased activity of this enzyme and defective oxidative phosphorylation.13 Thus, the phosphorylation status of PDH may serve as an index for the turnover of tricarboxylic acid cycle and ATP synthesis. In the present studies, ISO stimulation was also associated with a significant transient increase (≈60%) in the phosphorylation level of SCS and sustained depression (≈60%) of Complex I phosphorylation. Although the catalytic and regulatory subunits of cAMP-dependent PKA have been shown to exist in the inner membrane and matrix of bovine heart mitochondria,14 little is known about the physiological significance of the cAMP-dependent phosphorylation of SCS and complex I in the mammalian heart. The present studies represent the first evidence for β-adrenergic–induced modification of phosphorylation state of SCS and complex I in mouse cardiomyocytes.
Heat Shock Proteins
Most importantly, the present findings manifested de novo phosphorylation of a cardiac isoform of p20 in myocytes after prolonged (30 minutes or longer) ISO stimulation. Sequence analysis revealed high amino acid similarity, between the mouse cardiac p20 and the human and rat slow-twitch skeletal muscle p20,15,16 which is a heat shock–related protein and can be phosphorylated by the cAMP-PKA pathway.17 Interestingly, phosphorylation of p20 was also induced by insulin in skeletal muscle,18 which might be associated with insulin resistance. It is noteworthy that adenovirus-mediated overexpression of the cardiac p20 in adult rat cardiomyocytes increased cell contractility and intracellular Ca2+ transient amplitudes, indicating that p20 is involved in the regulation of myocardial contractility. This notion is further substantiated by the finding that, in rat slow-twitch skeletal muscle, the expression level of p20, probably a skeletal analogue of p20, is associated with muscle contraction.16 Because the cardiac p20 also contains the conserved domain sequence for heat shock proteins and the RRAS consensus sequence for cAMP-PKA phosphorylation substrates, we hypothesize that p20 belongs to the family of small heat shock proteins, and that its Ser16 phosphorylation via the cAMP-PKA pathway may play an important role in the modulation of cardiac function and remodeling, in response to sustained β-adrenergic stimulation, such as increased circulating catecholamine levels in heart failure.
In addition, we demonstrated transient and modest decreases in the phosphorylation levels of HSP27 and αB-crystallin during acute ISO stimulation (for 5 minutes). Numerous reports have shown that heat shock proteins may play multiple roles in cellular structure, metabolism, and adaptation to stress, thus protecting cells against various stimuli.19,20 Interestingly, the onset of heart failure in rats, after coronary artery ligation, was associated with increases in myocardial HSP27,21 whereas ischemic preconditioning caused translocation and phosphorylation of αB-crystallin in the isolated rat heart, suggesting its potential cardioprotective role.22 Furthermore, αB-crystallin was shown to inhibit both the mitochondrial and death receptor pathways mediated cell apoptosis.23 Thus, the decreased phosphorylation of HSP27 and αB-crystallin might contribute to β-adrenergic–induced cardiomyocyte apoptosis.7,24,25
In conclusion, 2-D gel electrophoresis in combination with 32P autoradiography enabled us to detect a large number of phosphoproteins concurrently, thus providing a novel and sensitive platform to analyze cardiac phosphoproteome and its modifications in response to various stimuli. Brief or sustained β-agonist stimulation resulted in significant but differential modification of the cardiomyocyte phosphoproteome, suggesting dynamic regulation of β-adrenergic signaling in the mammalian heart. Furthermore, a de novo phosphorylation of a cardiac heat shock protein p20 was identified, for the first time, in mouse cardiomyocytes, after prolonged activation of the β-adrenergic signaling pathway. Phosphorylation of the cardiac p20 occurs at Ser16 on β-agonist stimulation. The expression and phosphorylation of p20 appears to be involved in the regulation of cardiac contractility and Ca2+ handling. The potential role of p20 phosphorylation in β-adrenergic modulation of cardiac cell survival, cell death, and remodeling merits further investigation.
This work was supported by NIH grants HL-26057, HL-64018, HL-52318 (E.G.K.), and AG-12853 (J.E.M). We wish to thank Dr George Tsaprailis (University of Arizona), Brent Baldwin, and Qunying Yuan for their technical assistance with protein identification via HPLC tandem mass spectrometry, 2-D gel electrophoresis, and Western blotting, respectively.
Original received February 12, 2003; resubmission received June 19, 2003; revised resubmission received October 31, 2003; accepted November 6, 2003.
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