AJP - Heart AJP: Heart and Circulatory Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 293: H762-H769, 2007. First published April 6, 2007; doi:10.1152/ajpheart.00104.2007
0363-6135/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/1/H762    most recent
00104.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (10)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rodriguez, P.
Right arrow Articles by Kranias, E. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rodriguez, P.
Right arrow Articles by Kranias, E. G.

Phosphorylation of human inhibitor-1 at Ser67 and/or Thr75 attenuates stimulatory effects of protein kinase A signaling in cardiac myocytes

Patricia Rodriguez,1 Bryan Mitton,1 Persoulla Nicolaou,1 Guoli Chen,1 and Evangelia G. Kranias1,2

1Department of Pharmacology and Cell Biophysics, College of Medicine, University of Cincinnati, Cincinnati, Ohio; and 2Molecular Biology Division, Center for Basic Research, Foundation for Biomedical Research of the Academy of Athens, Athens, Greece

Submitted 25 January 2007 ; accepted in final form 1 April 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The depressed function of failing hearts has been partially attributed to increased protein phosphatase-1 through its impaired regulation by inhibitor-1. Phosphorylation of inhibitor-1 at Thr35 by PKA results in potent inhibition of protein phosphatase-1 activity, while phosphorylation at Ser67 or Thr75 by PKC attenuates the inhibitory activity. To examine the functional role of dual-site (Ser67, Thr75) phosphorylation of inhibitor-1 by PKC, the constitutively phosphorylated Ser67 (S67D) and/or Thr75 (T75D) human inhibitor-1 forms were expressed in adult cardiomyocytes. Expression of either single or double phosphorylated inhibitor-1 was associated with similar decreases in cardiac contractility, indicating that maximal inhibition can be elicited by each of these sites alone and that their inhibitory effects are not additive. Notably, activation of the cAMP pathway could only partially reverse the depressed contractile parameters. Accordingly, protein phosphatase-1 activity remained elevated, phosphorylation of phospholamban at Ser16 was decreased, and the EC50 values of the sarcoplasmic reticulum calcium transport system were higher compared with controls. Thus phosphorylation of Ser67 and/or Thr75 in inhibitor-1 may mitigate the stimulatory effects of the cAMP pathway, resulting in compromised cardiac function.

cardiac function; sarcoplasmic reticulum; contractility; calcium cycling

MAINTENANCE OF PROPER HEART function requires fine-tuning of the orchestration of intracellular signaling pathways, which regulate complex processes such as contractility, hypertrophy, and cell death. The precise regulation of kinases and phosphatases is crucial to these processes. In the context of the failing heart, numerous disruptions in the kinase/phosphatase balance have been shown to render the cardiomyocyte unable to adequately cope with stress. Among these, attenuation of signaling through the beta-adrenergic receptor/cAMP pathway, which may occur as a result of receptor downregulation, receptor desensitization, and receptor uncoupling, is a primary insult (1, 10). This alteration is associated with reduced PKA phosphorylation of intracellular targets, such as phospholamban, and concomitantly reduced cardiac contractility (7, 18). Importantly, the activity of protein phosphatase-1 (PP1) is increased in the failing heart, which further enables dephosphorylation of important regulatory phosphoproteins leading to an overall decreased function (6). In addition to disrupted PKA and PP1 regulation, an increase in the levels of select PKC isoforms ({alpha}, beta, and {epsilon}) also leads to disrupted PKC signaling pathways (3, 13). Intriguingly, animal models have revealed that the levels of PKC-{alpha} in particular appear inversely related to cardiac function. Transgenic overexpression of PKC-{alpha} was associated with reduced cardiac contractility, while PKC-{alpha}-null hearts were hypercontractile (4). Thus the detrimental effects of attenuated beta-adrenergic signaling in the failing heart may be compounded by the effects of increased PKC-{alpha} expression.

Importantly, these two signaling pathways converge on protein phosphatase inhibitor-1 (I-1), a key regulator of PP1 activity. Since PP1 opposes PKA signaling in the heart by dephosphorylating targets such as phospholamban, the ryanodine receptor, and troponin I, the regulatory role played by I-1 has been a major research focus. On phosphorylation of I-1 by PKA at Thr35, I-1 potently inhibits PP1, allowing PKA phosphorylation to propagate and increase cardiac contractility in an unopposed manner (6). Indeed, chronic inhibition of PP1 by a constitutively active form of I-1 (AA 1-65, T35D) resulted in tremendous gains in contractility (15). Although the role of inhibitor-1 as a PKA-stimulated inhibitor of PP1 has been well characterized, the functional consequence of phosphorylation of this protein at other sites is still a focus of investigation. Recently, it was found that I-1 is phosphorylated in cardiac muscle at Ser67 by PKC-{alpha}, resulting in an indirect effect in the regulation of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a pump (4), but the precise mechanism involved in this signaling pathway is not yet fully understood. Evidence supports the concept that phosphorylation of I-1 by PKC-{alpha} leads to an increase in PP1 activity (4). Accordingly, our recent studies (16) identified a new in vitro PKC-{alpha} phosphorylation site in human I-1 at Thr75, which caused a direct increase in PP1 activity and depressed myocyte mechanical function.

Elucidating the interplay between these two newly identified phosphorylation sites on I-1 function and PP1 activity is of great importance, since PP1 activity appears to be a fundamental determinant of cardiac performance. The role of I-1 in the heart becomes especially salient in the context of heart failure, as evidenced by studies of several animal models in which increases in PP1 activity similar to those observed in human failing hearts result in severe cardiac decompensation, failure, and premature death (6, 14).

Thus, to better understand the mechanisms by which the Ser67 and Thr75 phosphorylations of I-1 regulate cardiac contractility, we have expressed constitutively phosphorylated I-1 mutants in adult cardiomyocytes. We show that prior phosphorylation of I-1 at these sites attenuates its ability to become phosphorylated by the cAMP pathway and reduces the extent to which PP1 activity is inhibited, resulting in diminished SERCA2a transport function, decreased phospholamban phosphorylation at Ser16, and cardiac contractility. These findings offer new insights into the mechanisms underlying regulation of PP1 in cardiac cells.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. PKA catalytic subunit and cAMP were purchased from Upstate Biotechnology. The pGEX 6P-3 plasmid, glutathione Sepharose 4B, and PreScission protease were obtained from Amersham Biosciences. Quik-Change II site-directed mutagenesis kits and BL21 CodonPlus (DE3)-RIPL competent cells were obtained from Stratagene. Diacylglycerol, ampicillin, isopropyl-1-thio-beta-D-galactopyranosidase, protease inhibitor cocktail, and forskolin were obtained from Sigma-Aldrich. T4 ligase and EcoRI and NotI restriction enzymes were purchased from New England Biolabs. [{gamma}-32P]ATP was obtained from Perkin Elmer. Laminin was from BD Biosciences. Phorbol 12,13-dibutyrate (PDBu) was purchased from Calbiochem. AC1 is a custom-made (Affinity Bioreagents) rabbit polyclonal affinity-purified antibody against the NH2-terminal sequence of I-1 (residues 1–15). Anti-phospholamban and anti-glutathione S-transferase (GST) antibodies were obtained from Affinity Bioreagents. Antibody against phospholamban phosphorylated at Ser16 was purchased from Badrilla. Mouse monoclonal antibody against PP1 was from Santa Cruz Biotechnology.

Generation of I-1 recombinant proteins and I-1 adenoviruses. The human I-1 cDNA (NCBI Accession No. CK823634) was a gift from Dr. Shirish Shenolikar (Duke University, Durham, NC). I-1 cDNA was cloned into the pGEX-6P-3 plasmid and expressed as a GST-fusion protein in BL21 CodonPlus (DE3)-RIPL competent cells, as described previously (17). Briefly, fusion proteins were purified, and the GST tag was cleaved and removed from the medium. Samples were analyzed by SDS-PAGE to estimate the extent of cleavage and protein yield after purification. Protein concentration was determined by Micro BCA assay (Pierce). The I-1(S67D/T75D) mutant was derived from the I-1 wild-type (I-1WT) sequence with site-directed mutagenesis in a stepwise fashion (Fig. 1). Adenoviruses encoding green fluorescent protein (GFP) (Ad.GFP), I-1WT (Ad.I-1WT), or I-1 mutants I-1(S67D), I-1(T75D), or I-1(S67D/T75D) [Ad.I-1(S67D), Ad.I-1(T75D), Ad.I-1(S67D/T75D)] were generated by using the Ad-Easy XL system in Ad-293 cells. The Quik-Change Site-Directed Mutagenesis II kit (Stratagene) was used to introduce mutations into the original I-1 cDNA. The viruses were purified with the Adenovirus Mini Purification Kit (Virapur) and titered with the Adeno-X Rapid Titer Kit (Clontech), as previously described (15) (Fig. 1).


Figure 1
View larger version (27K):
[in this window]
[in a new window]

  		Fig. 1. Schematic diagram of inhibitor-1 (I-1) recombinant proteins and adenoviral vectors. The human I-1 cDNA was cloned into the pGEX-6P3 vector for expression as a glutathione S-transferase (GST)-fusion protein. For generation of recombinant adenoviruses, the cDNAs were subcloned from the pGEX-6P-3 vector into the pShuttle-IRES-hrGFP-1 vector and inserted into the AdEasy-1 viral backbone by homologous recombination. The mutations shown were introduced into the original cDNA by site-directed mutagenesis. CMVp, cytomegalovirus promoter; GFP, green fluorescent protein; WT, wild type.

 
Primary culture of rat isolated ventricular cardiomyocytes, adenovirus infection, and contractile parameter measurements. Ventricular myocytes from adult male Sprague-Dawley rats ({approx}300 g) were isolated by collagenase digestion as previously detailed (17). Rats were handled according to a protocol approved by the Institutional Animal Care and Use Committee at the University of Cincinnati. Plated myocytes were infected with adenoviruses at a multiplicity of infection of 500 for 2 h at 37°C in a humidified 5% CO2 incubator. After 24 h of infection, myocyte contraction at the basal level and under forskolin (100 nM) was performed at room temperature by using a Grass S5 stimulator (0.5 Hz). Fractional shortening (FS) and maximal rates of contraction and relaxation (dL/dtmax) were calculated with a video edge motion detector (Crescent Electronics). For immunoblotting, sarcoplasmic reticulum (SR) Ca2+ uptakes, and protein phosphatase activity assays, cultured infected cardiomyocytes were harvested and homogenized with a Polytron in solubilization buffer containing (mM) 50 Tris·HCl (pH 7.0), 10 NaF, 1 EDTA, 0.3 sucrose, 0.3 PMSF, and 0.5 mM DL-dithiothreitol, with protease inhibitor cocktail (1 ml/20 g tissue). NaF was omitted from the buffer for determination of protein phosphatase activity. For PKA activation, infected myocytes were treated with forskolin (100 nM) for 15 min before homogenization and subsequent Ca2+ uptake or protein phosphatase activity assays. For PKC activation, cells were treated with PDBu (1.5, 2.5, and 3.5 µM) for 10 min (17) before contractility was recorded.

SR Ca2+ uptake in cultured rat cardiomyocytes. After 24-h infection of isolated rat cardiomyocytes, cells were harvested and homogenized at 4°C in the buffer described above. The initial rates of SR Ca2+ uptake were determined in myocyte homogenates with the Millipore filtration technique and 45CaCl2 over a range of Ca2+ concentrations (pCa 5–8). Ca2+ uptake into cardiomyocytes was initiated by addition of 5 mM ATP, and aliquots were filtered through a 0.45-µm Millipore filter after 0, 30, 60, and 90 s to terminate the reaction, as previously detailed (17). The specific Ca2+ uptake values [maximum Ca2+ uptake rate (Vmax) and EC50] were analyzed with the OriginLab 5.1 program.

Protein phosphatase activity assay. Protein phosphatase activity was assessed in infected cardiomyocyte homogenates (1 µg) with the Protein Serine/Threonine Phosphatase Assay System (New England Biolabs) according to the manufacturer's instructions and as described previously (16). Okadaic acid (10 nM) was used to discern between PP1 and protein phosphatase-2A (PP2A) activities (14, 16).

PKA in vitro phosphorylation assays. Recombinant I-1 or I-1(S67D/T75D) mutant (7 µg) was phosphorylated by PKA catalytic subunit (0.1 µg) at 35°C in (mM) 50 Tris·HCl (pH 7.0), 5 MgCl2, 5 NaF, and 1 EGTA, with 1 µM cAMP (included as an extra precaution although the PKA catalytic subunit was used) and 0.25 mM [{gamma}-32P]ATP (0.4 µCi/nmol). After 1 h, the reactions were stopped by adding SDS sample buffer to the medium. For the control samples, cAMP and PKA were omitted from the buffer. The amount of [32P]phosphate incorporated into I-1 proteins was determined by SDS-PAGE and autoradiography. Densitometric analysis of the data was conducted with ImageQuant 5.2 software.

Immunoblot analysis. Proteins were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes. After 1–2 h in 5% dried milk, membranes were probed overnight at 4°C with primary antibodies. A secondary peroxidase-labeled antibody (Amersham Biosciences) was used in combination with an enhanced chemiluminescent detection system (Supersignal West Pico Chemiluminescent, Pierce) to visualize the primary antibodies. The optical density of the bands was analyzed by ImageQuant 5.2 software.

Statistics. All values are expressed as means ± SE for n experiments. Comparisons were evaluated by Student's t-test for unpaired data or one-way ANOVA, as appropriate.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of constitutively phosphorylated I-1 at Ser67 and/or Thr75 on myocyte contractility and SR Ca2+ uptake. We showed previously (16) that phosphorylation of I-1 at Thr75 by PKC-{alpha} induces a significant decrease in cardiomyocyte performance. Since PKC-{alpha} also phosphorylates Ser67 in cardiac muscle (4), we explored whether phosphorylation of this site has the same effects on myocyte contractility as phosphorylation of Thr75, and if so, whether dual-site phosphorylation may have additive or even synergistic effects. To achieve this, adult rat cardiomyocytes were infected with adenoviruses expressing I-1 wild type (Ad.I-1WT), single-site constitutively phosphorylated I-1 [Ad.I-1(S67D) or Ad.I-1(T75D)], or dual-site phosphorylated I-1 [Ad.I-1(S67D/T75D)]. An adenovirus expressing GFP (Ad.GFP) was used as control. Adenoviral infection efficiency, assessed by green fluorescence and Western blot immunodetection, indicated that the levels of wild-type or mutant I-1 expression were similar in all groups (Fig. 2A), whereas endogenous I-1 was undetectable in cells infected with Ad.GFP, similar to previous observations (8, 16). Infection with Ad.I-1WT had no effects on basal contractile parameters, while expression of I-1(S67D) reduced the rates of myocyte contraction (22%) and relaxation (27%), as well as FS (25%) to a similar extent as myocytes expressing I-1(T75D) (Fig. 2B). Although the functional performance of myocytes expressing I-1(T75D) tended to be more attenuated than those expressing I-1(S67D), the values were not significantly different. Interestingly, expression of the constitutively dual-site phosphorylated (S67D and T75D) I-1 yielded decreases in the maximal velocities of contraction and relaxation as well as FS similar to those elicited by the single mutants (Fig. 2B). These results indicate that phosphorylation of either Ser67 or Thr75 in I-1 results in similar decreases on myocyte contractility and that simultaneous phosphorylation of both sites does not increase these inhibitory effects.


Figure 2
View larger version (49K):
[in this window]
[in a new window]

  		Fig. 2. Phosphorylation of I-1 at Ser67 and/or Thr75 depresses myocyte cardiac function and sarcoplasmic reticulum (SR) Ca2+ uptake. A: adult rat cardiomyocyte 24 h after adenoviral infection at a multiplicity of infection of 500. Bottom: green fluorescence protein (GFP) expression. A custom-made antibody specific for I-1 (AC1; 1:1,000) was used to detect overexpression of the protein in myocytes infected with GFP (lane 1) I-1WT (lane 2), or I-1 mutants I-1(S67D) (lane 3), I-1(T75D) (lane 4), or I-1(S67D/T75D) (lane 5). Top: staining with Coomassie blue to demonstrate equal protein loading. B: maximal rates of contraction and relaxation (dL/dtmax), fractional shortening (FS), and average EC50 values of adenovirus-infected cardiomyocytes under basal conditions; 15–20 myocytes/heart were analyzed with a total number of hearts per group of 12 (Ad.GFP), 6 (Ad.I-1WT), 6 [Ad.I-1(S67D)], 8 [Ad. I-1(T75D)], or 6 [Ad.I-1(S67D/T75D)]. *P < 0.05, **P < 0.01; ***P < 0.001, comparison of all groups vs. GFP. #P < 0.05, comparison among the 3 mutants.

 
It has been previously described that PKC-{alpha} phosphorylates I-1 at Ser67 and Thr75 (4, 17). Therefore, to explore whether the negative inotropic effect of I-1(S67D) or I-1(T75D) is elicited by PKC-mediated phosphorylation of I-1, myocytes expressing I-1WT or I-1(S67D/T75D) were treated with several concentrations of the PKC activator PDBu. PDBu induced a significant decrease in FS in cells expressing I-1WT, which was dose dependent (Fig. 3). However, there was no further decrease in the contractility of myocytes infected with the I-1(67D/75D) mutant, suggesting that PKC phosphorylation of I-1 may be partially mediating the depressive effects induced by phorbol esters on cardiomyocyte contractile parameters.


Figure 3
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 3. Effects of PKC activation by phorbol 12,13-dibutyrate (PDBu) on contractility of myocytes expressing I-1WT and the double I-1 mutant. FS of adenovirus-infected myocytes with I-1WT or I-1(S67D/T75D) mutant, under basal conditions or with PDBu treatment at several concentrations (1.5, 2.5, or 3.5 µM) is shown; 15–20 myocytes/heart were analyzed from 3 hearts. B, basal conditions. *P < 0.05, comparison of basal vs. PDBu in I-1WT group. $P < 0.05, comparison of I-1WT vs. I-1(S67D/T75D) at basal conditions. Note that there were no significant differences between I-1WT and I-1(S67D/T75D) with PDBu treatment.

 
The depressed contractility of the failing heart is thought to involve alterations in the SR Ca2+ cycling that, at least in part, may be associated with depressed SR Ca2+ uptake (19). Consistent with this notion, we examined SERCA2a function in myocytes infected with I-1 constitutively phosphorylated at Ser67 and/or Thr75. The initial rates of Ca2+ transport were measured over a wide range of Ca2+ concentrations, similar to those present in vivo during relaxation and contraction. Homogenates generated from myocytes infected with adenoviruses expressing GFP, as a control, and the I-1 forms I-1WT, I-1(S67D), I-1(T75D), or I-1(S67D/T75D) were processed under conditions that restrict Ca2+ uptake to SR (12). Infection with Ad.GFP was associated with an EC50 value of SERCA2a for Ca2+ of 0.294 ± 0.01 µM. This value was similar to that exhibited by I-1WT-overexpressing myocytes (0.336 ± 0.013 µM; Fig. 2B). However, the apparent EC50 of SERCA2a for Ca2+ was significantly increased in myocytes expressing the I-1(S67D), I-1(T75D), or I-1(S67D/T75D) mutant (0.457 ± 0.012, 0.664 ± 0.014, and 0.611 ± 0.005 µM, respectively; Fig. 2B), similar to contractile parameters.

Effects of PKA activation on contractility in myocytes expressing phospho-Ser67 and/or phospho-Thr75 I-1. Activation of the beta-adrenergic/cAMP pathway in the heart results in enhanced function, which may overcome the depressive effects of proteins that inhibit cardiac contractility (11). To assess whether stimulation of this signaling pathway is capable of reversing the impaired function of myocytes expressing constitutively phosphorylated I-1 mutants, adenovirus-infected cardiomyocytes were treated with a range of forskolin concentrations from 10 nM to 1 µM. Surprisingly, high doses of forskolin elicited arrhythmias in myocytes expressing constitutively phosphorylated Ser67 and/or Thr75 I-1, but not in I-1 WT- or GFP-expressing cells. Therefore, we selected 0.1 µM, which appeared optimal in GFP or I-1WT-infected myocytes, as the highest forskolin concentration that induced stimulation of contractility without eliciting arrhythmias. Forskolin treatment of myocytes expressing GFP caused dramatic increases in the velocities of contraction (38%) and relaxation (51%), as well as in FS (8.5%), compared with basal levels (Fig. 4). The increases in performance of cells expressing I-1WT were similar to the control cells (36.5%, 49%, and 15.5% for the rate of contraction, rate of relaxation, and FS, respectively). Importantly, cardiac function of myocytes infected with Ad.I-1(S67D), Ad.I-1(T75D), or Ad.I-1(S67D/T75D) also improved on forskolin treatment, but the cardiac parameters did not reach the maximal levels observed in either the Ad.I-1WT or Ad.GFP groups (Fig. 4). However, the relative increases in the contractile parameters of the forskolin-stimulated cardiomyocytes expressing the constitutively phosphorylated I-1 mutants were similar to the increases obtained by the Ad.GFP and Ad.I-1WT infections, indicating no alterations in the upstream PKA signaling pathway. These data suggest that although cardiac contractility of myocytes expressing the phosphorylated I-1 mutants can be enhanced by forskolin treatment, the overall function remains depressed compared with controls.


Figure 4
View larger version (22K):
[in this window]
[in a new window]

  		Fig. 4. Effect of PKA activation on the contractility of myocytes infected with Ad.I-1(S67D), Ad.I-1(T75D), and Ad.I-1(S67D/T75D). FS and maximal rates of contraction and relaxation (dL/dtmax) of adenovirus-infected cardiomyocytes treated with 0.1 µM forskolin are shown. Total number of hearts: 10 (Ad.GFP), 6 (Ad.I-1WT), 6 [Ad.I-1(S67D)], 6 [Ad.I-1(T75D)], and 6 [Ad.I-1(S67D/T75D)] with 15–20 myocytes/heart. *P < 0.05; **P < 0.01.

 
Effects of PKA activation on SR Ca2+ uptake in adenovirus-infected myocytes. To explore whether the impaired SERCA2a transport function observed in cardiomyocytes expressing I-1 phosphorylated at Ser67 and/or Thr75 may be reversed by activation of the cAMP signaling pathway, adenovirus-infected cells were treated with forskolin. As expected, the EC50 values under PKA stimulation were reduced in myocytes expressing GFP, I-1WT, or I-1(S67D), I-1(T75D), or I-1(S67D/T75D). Importantly, the relative decreases were similar in all five groups, compared with their corresponding basal values (Figs. 2B and 5, A and B). Analysis of the corresponding values of the Ca2+ transport system on PKA stimulation indicated that cardiomyocytes expressing GFP or I-1WT had similar EC50 parameters (0.17 ± 0.029 and 0.147 ± 0.005 µM, respectively), and these were the most stimulated parameters among the five groups (Fig. 5B). Although forskolin also decreased the EC50 values in cardiomyocytes expressing the constitutively phosphorylated I-1 mutant at Ser67 (0.234 ± 0.005 µM), Thr75 (0.342 ± 0.016 µM), or both sites (0.334 ± 0.053 µM) compared with their respective basal values (Fig. 5B), the Ca2+ uptake rates remained depressed in the mutant-expressing cells compared with myocytes expressing either I-1WT or GFP (Fig. 5, A and B). There were no statistically significant differences among the I-1 mutants' EC50 values (Fig. 5B). Notably, stimulation of PKA in these groups improved SR Ca2+ uptake values only to the basal levels of the control cells. There were no alterations in the maximum velocities (Vmax) of Ca2+ uptakes among the five groups. Interestingly, phosphorylation of phospholamban, the regulator of SERCA2a function, was reduced by 35% at Ser16 in myocytes expressing the I-1 mutants compared with the GFP or I-1WT groups on PKA stimulation (Fig. 5C). Additionally, SERCA2a protein levels were similar among the five groups (Fig. 5C). These results indicate that phosphorylation of I-1 at either Ser67 and/or Thr75 is associated with attenuated stimulation of SERCA2a transport, which may be, at least in part, due to reduced phospholamban phosphorylation at Ser16.


Figure 5
View larger version (32K):
[in this window]
[in a new window]

  		Fig. 5. Effect of PKA stimulation on the Ca2+ affinity of sarcoplasmic reticulum (SR) Ca2+ transport of myocytes expressing I-1 phosphorylated at Ser67 and/or Thr75. The initial rates of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a Ca2+ transport were assessed in cardiomyocytes infected with Ad.GFP, Ad.I-1WT, Ad.I-1(S67D), Ad.I-1(T75D), and Ad.I-1(S67D/T75D) with forskolin treatment. A: data were normalized to the calculated Vmax for each group and fit to a sigmoidal curve by using the OriginLab 5.1 program. Symbols represent the average of 3 independent experiments, assayed in duplicate. B: average EC50 values for each group: statistical comparisons of each group vs. GFP. C: representative blots showing phosphorylation of phospholamban (PLN) at Ser16, using phosphospecific antibodies (1:5,000; Badrilla). The same membranes were striped and probed for total PLN (1:1,000; Affinity Bioreagents) and SERCA2a (1:1,000; custom-made antibody, Affinity Bioreagents). A total of 4 independent experiments were used for statistical analysis. *P < 0.05; **P < 0.01.

 
PP1 activity in adenovirus-infected cardiomyocytes treated with forskolin. Since PP1 is regulated by I-1, which acts as an inhibitor on PKA stimulation, it was of special interest to explore whether phosphorylation of I-1 at Ser67 and/or Thr75 may affect PP1 activity after stimulation with forskolin. The degree of total protein phosphatase inhibition after PKA stimulation was similar among the GFP and mutant I-1-infected groups. However, the myocytes expressing I-1WT exhibited a significantly higher inhibition compared with the GFP group, indicating that the endogenous levels of I-1 may be limiting and that overexpression of this molecule results in increased inhibition of PP1 activity (Fig. 6). Accordingly, selective PP1 inhibition, assessed by using 10 nM okadaic acid as a PP2A inhibitor (14, 16), showed that myocytes expressing I-1WT inhibited PP1 activity more potently under forskolin, compared with the GFP control. Interestingly, this increased inhibition of PP1 was not associated with any significant alterations in phospholamban phosphorylation, the EC50 of SERCA2a (Fig. 5B), or contractility (Fig. 4), suggesting that maximal stimulation appears to occur by phosphorylation of the endogenous I-1 under our conditions. In contrast, the three constitutively phosphorylated I-1 mutants, I-1(S67D), I-1(T75D), and I-1(S67D/T75D), presented less inhibition of PP1, compared with either GFP control or I-1WT-infected cells. Consistent with contractility assays (Fig. 4), the degree of PP1 inhibition in myocytes expressing the I-1 double mutant [I(S67D/T75D)] was similar to that exhibited by either I-1(S67D) or I-1(T75D), indicating that simultaneous phosphorylation of the two sites did not exhibit an additive effect in the inhibition of PP1 activity after PKA stimulation. Similar to previous observations (16), PP1 protein levels were not altered in any of the groups (data not shown).


Figure 6
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 6. Percentage of inhibition of protein phosphatase-1 (PP1) activities in adenovirus-infected myocytes on PKA stimulation. Total phosphatase (PP) activity was assayed in forskolin-treated myocytes lysates expressing Ad.GFP, Ad.I-1WT, Ad.I-1(S67D), Ad.I-1(T75D), and Ad.I-1(S67D/T75D). Okadaic acid (10 nM) was added to cell lysates to differentiate type 1 and 2A phosphatase activities. Bars represent the average of 3 independent myocyte lysates assayed per duplicate. *P < 0.05 represents comparison of each group vs. GFP.

 
Phosphorylation of I-1 at Ser67 and Thr75 reduces PKA-dependent phosphorylation. To better understand the mechanism by which I-1 phosphorylated at Ser67/Thr75 is a poor inhibitor of PP1 activity, we analyzed whether these phosphorylations reduce the ability of PKA to phosphorylate I-1 at Thr35. It has been reported that substitution of Thr35 on I-1 by an alanine completely abolishes I-1 phosphorylation by PKA (9). Moreover, this mutated I-1 failed to inhibit PP1 activity, further establishing that Thr35 is the PKA site in I-1 and that it is the only site associated with phosphatase inhibitory activity. Therefore, we examined the extent of incorporation of radioactivity after PKA phosphorylation of recombinant I-1WT and I-1(S67D/T75D) proteins in the presence of [{gamma}-32P]ATP. As shown in Fig. 7, phosphorylation of Thr35 in the double mutant was reduced by 29% compared with I-1WT. However, the introduction of the false amino acids in the recombinant protein may affect phosphorylation by PKA. Therefore, the inhibition of PP1 activity was compared in 1) I-1WT recombinant protein, which was first phosphorylated by PKC-{alpha} in vitro and subsequently phosphorylated by PKA, and 2) I-1(S67D/T75D) recombinant protein. The percentage of PP1 inhibition (12.2 ± 1.7%) was similar to I-1(S67D/T75D) protein phosphorylated by PKA (11.8 + 4.5%) (data not shown), indicating that the reduced PKA phosphorylation of the mutant may not be due to the introduction of the false amino acids. Taken together, these results suggest that phosphorylation of I-1 at Ser67 and Thr75 reduces its subsequent phosphorylation by PKA, which may contribute to depressed phospholamban phosphorylation at Ser16 (Fig. 5C), SERCA2a Ca2+ transport (Fig. 5B), and contractility (Fig. 4) on PKA stimulation.


Figure 7
View larger version (33K):
[in this window]
[in a new window]

  		Fig. 7. PKA in vitro phosphorylation of recombinant I-1WT and I-1(S67D/T75D) mutant. A: radiolabeled phosphoproteins were detected by autoradiography. B: same membrane was probed with a specific custom-made antibody for I-1 (AC1; 1:1,000). C, control samples; PKA, phosphorylated samples. C: radioactivity associated with I-1 was quantified by densitometry, corrected for background. Bars show average of 3 independent phosphorylation reactions. *P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Heart failure is a condition of multiple etiologies associated with, among others, desensitization of the beta-adrenergic pathway in response to chronically increased sympathetic tone (5) and increased expression of the Ca2+-dependent PKC-{alpha} isoform (3). These initial compensatory adaptations may ultimately lead to detrimental consequences in the heart. A regulatory phosphoprotein that integrates both the PKA (8) and PKC-{alpha} (4, 16) signaling pathways is protein phosphatase inhibitor-1 (I-1). The positive effects of PKA phosphorylation of I-1 in the heart have been well documented (6, 15), but the precise role of the PKC phosphorylation sites in I-1 and their interaction with the cAMP-dependent kinase pathway have not been delineated, and they were the focus of this study.

Several lines of evidence from human and animal models of heart failure have established that the activity of PP1 is inversely related to cardiac performance (6, 14). Attenuation of beta-adrenergic signaling may underlie these effects in part, since the normal balance of PKA and PP1 activities would shift in favor of PP1 in this context. Additionally, it has been postulated that the increased PP1 activity in failing hearts is associated with diminished cAMP-dependent phosphorylation and activation of I-1 (8). Consistent with this notion, a new emerging pathway in the regulation of PP1 suggests that phosphorylation of I-1 by PKC-{alpha} may contribute to enhanced PP1 activity (4, 16). Indeed, our recent study (16), which identified Thr75 as a new PKC-{alpha} site in I-1, showed that T75D I-1 recombinant protein induced an increased of PP1 activity. Accordingly, we found that constitutive phosphorylation of I-1 at Ser67 and/or Thr75 significantly reduces the extent to which PP1 becomes inhibited after PKA stimulation in cardiomyocytes. Importantly, contractility was also diminished under basal conditions. This observation was unexpected, since PKA basal activity is thought to be low in unstimulated cardiomyocytes, implying additional pathways that mediate the depressive effects of the I-1 mutants on basal contractility.

Interestingly, the two sites appeared equivalent, and no additive effect was observed when both sites were simultaneously phosphorylated. These results raise the possibility that phosphorylations of Ser67 and Thr75 may exert their effects independently from each other. However, it remains unclear whether phosphorylation of both targets occurs simultaneously or whether both sites are maximally phosphorylated in vivo. As such, phosphorylation of I-1 at both sites would be required to elicit maximal response, explaining the nonadditive influence of phospho-Ser67 and phospho-Thr75 observed in the present study.

The poor inhibition of PP1 activity on PKA activation in myocytes expressing phospho-Ser67 and phospho-Thr75 I-1 may be due to the decreased ability of I-1 to become phosphorylated by PKA, as indicated by our in vitro findings. These results are in agreement with a recent report, which showed that phosphorylation of Ser65 and Ser67 altered the ability of I-1 to serve as a PKA substrate in mouse brain tissue (17).

It has been shown that transgenic mice either lacking or overexpressing PKC-{alpha} present enhanced or reduced cardiac contractility, respectively. Moreover, modulation of PKC-{alpha} activity in those mouse models affected phosphorylation of phospholamban (4). Accordingly, constitutive phosphorylation of I-1 at Ser67 and/or Thr75 was associated with decreased phospholamban phosphorylation at Ser16 and depressed SR Ca2+ uptake rates under PKA activation. Consequently, the mechanical performance of the cardiomyocytes expressing I-1 mutants mirrored SR Ca2+ uptake measurements. Furthermore, PKA activation by forskolin did not improve SERCA2a function or contractility to the same extent as myocytes expressing I-1WT or GFP. Therefore, we postulate that the poor inhibition of PP1 in cardiomyocytes expressing the PKC-phosphorylated I-1 on PKA activation may lead to reduced phospho-Ser16 phospholamban, which impairs SERCA2a function and leads to depressed myocyte contractility. These findings suggest an important cross talk between PKA and PKC through I-1.

Taken together, our data suggest that enhanced PKC signaling in failing hearts may result in increased phosphorylation of I-1 at Ser67 and Thr75. These phosphorylations may work to partially suppress the beta-adrenergic signaling cascade and consequently reduce the stimulatory effects on contractility through the maintenance of an abnormally enhanced PP1 activity. Thus I-1 appears to regulate cardiac contractility, and targeted regulation of its activity may be beneficial in the deteriorated function of failing hearts.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-26057, HL-64018, and HL-77101 and Leducq Fondation (to E. G. Kranias). P. Rodriguez is a recipient of a Programme 3+3 fellowship from the Centro Nacional de Investigaciones Cardiovasculares Carlos III, Spain.


   ACKNOWLEDGMENTS
 
We thank Sarah E. K. Figueira for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. G. Kranias, Dept. of Pharmacology and Cell Biophysics, Univ. of Cincinnati, Coll. of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0575 (e-mail: kraniaeg{at}email.uc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Barki-Harrington L, Perrino C, Rockman HA. Network integration of the adrenergic system in cardiac hypertrophy. Cardiovasc Res 63: 391–402, 2004.[Abstract/Free Full Text]
  2. Bibb JA, Nishi A, O'Callaghan JP, Ule J, Lan M, Snyder GL, Horiuchi A, Saito T, Hisanaga S, Czernik AJ, Nairn AC, Greengard P. Phosphorylation of protein phosphatase inhibitor-1 by Cdk5. J Biol Chem 276: 14490–14497, 2001.[Abstract/Free Full Text]
  3. Bowling N, Walsh RA, Song G, Estridge T, Sandusky GE, Fouts RL, Mintze K, Pickard T, Roden R, Bristow MR, Sabbah HN, Mizrahi JL, Gromo G, King GL, Vlahos CJ. Increased protein kinase C activity and expression of Ca2+-sensitive isoforms in the failing human heart. Circulation 99: 384–391, 1999.[Abstract/Free Full Text]
  4. Braz JC, Gregory K, Pathak A, Zhao W, Sahin B, Klevitsky R, Kimball TF, Lorenz JN, Nairn AC, Liggett SB, Bodi I, Wang S, Schwartz A, Lakatta EG, DePaoli-Roach AA, Robbins J, Hewett TE, Bibb JA, Westfall MV, Kranias EG, Molkentin JD. PKC-{alpha} regulates cardiac contractility and propensity toward heart failure. Nat Med 10: 248–254, 2004.[CrossRef][Web of Science][Medline]
  5. Bristow MR. Beta-adrenergic receptor blockade in chronic heart failure. Circulation 101: 558–569, 2000.[Free Full Text]
  6. Carr AN, Schmidt AG, Suzuki Y, del Monte F, Sato Y, Lanner C, Breeden K, Jing SL, Allen PB, Greengard P, Yatani A, Hoit BD, Grupp IL, Hajjar RJ, DePaoli-Roach AA, Kranias EG. Type 1 phosphatase, a negative regulator of cardiac function. Mol Cell Biol 22: 4124–4135, 2002.[Abstract/Free Full Text]
  7. Dash R, Kadambi V, Schmidt AG, Tepe NM, Biniakiewicz D, Gerst MJ, Canning AM, Abraham WT, Hoit BD, Liggett SB, Lorenz JN, Dorn GW 2nd, Kranias EG. Interactions between phospholamban and beta-adrenergic drive may lead to cardiomyopathy and early mortality. Circulation 103: 889–896, 2001.[Abstract/Free Full Text]
  8. El-Armouche A, Pamminger T, Ditz D, Zolk O, Eschenhagen T. Decreased protein and phosphorylation level of the protein phosphatase inhibitor-1 in failing human hearts. Cardiovasc Res 61: 87–93, 2004.[Abstract/Free Full Text]
  9. Endo S, Zhou X, Connor J, Wang B, Shenolikar S. Multiple structural elements define the specificity of recombinant human inhibitor-1 as a protein phosphatase-1 inhibitor. Biochemistry 35: 5220–5228, 1996.[CrossRef][Medline]
  10. Ferguson SS. Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Pharmacol Rev 53: 1–24, 2001.[Abstract/Free Full Text]
  11. Haghighi K, Schmidt AG, Hoit BD, Brittsan AG, Yatani A, Lester JW, Zhai J, Kimura Y, Dorn GW 2nd, MacLennan DH, Kranias EG. Superinhibition of sarcoplasmic reticulum function by phospholamban induces cardiac contractile failure. J Biol Chem 276: 24145–24152, 2001.[Abstract/Free Full Text]
  12. Kiss E, Ball NA, Kranias EG, Walsh RA. Differential changes in cardiac phospholamban and sarcoplasmic reticular Ca2+-ATPase protein levels. Effects on Ca2+ transport and mechanics in compensated pressure-overload hypertrophy and congestive heart failure. Circ Res 77: 759–764, 1995.[Abstract/Free Full Text]
  13. Liu X, Wang J, Takeda N, Binaglia L, Panagia V, Dhalla NS. Changes in cardiac protein kinase C activities and isozymes in streptozotocin-induced diabetes. Am J Physiol Endocrinol Metab 277: E798–E804, 1999.[Abstract/Free Full Text]
  14. Neumann J, Eschenhagen T, Jones LR, Linck B, Schmitz W, Schoolz H, Zimmermann N. Increased expression of cardiac phosphatases in patients with end-stage heart failure. J Mol Cell Cardiol 29: 265–272, 1997.[CrossRef][Web of Science][Medline]
  15. Pathak A, del Monte F, Zhao W, Schultz JE, Lorenz JN, Bodi I, Weiser D, Hahn H, Carr AN, Syed F, Mavila N, Jha L, Qian J, Marreez Y, Chen G, McGraw DW, Heist EK, Guerrero JL, DePaoli-Roach AA, Hajjar RJ, Kranias EG. Enhancement of cardiac function and suppression of heart failure progression by inhibition of protein phosphatase 1. Circ Res 15: 756–766, 2005.
  16. Rodriguez P, Mitton B, Waggoner JR, Kranias EG. Identification of a novel phosphorylation site in protein phosphatase inhibitor-1 as a negative regulator of cardiac function. J Biol Chem 281: 38599–38608, 2006.[Abstract/Free Full Text]
  17. Sahin B, Shu H, Fernandez J, El-Armouche A, Molkentin JD, Nairn AC, Bibb JA. Phosphorylation of protein phosphatase inhibitor-1 by protein kinase C. J Biol Chem 281: 24322–24335, 2006.[Abstract/Free Full Text]
  18. Schwinger RH, Munch G, Bolck B, Karczewski P, Krause EG, Erdmann E. Reduced Ca2+-sensitivity of SERCA 2a in failing human myocardium due to reduced serine-16 phospholamban phosphorylation. J Mol Cell Cardiol 31: 479–491, 1999.[CrossRef][Web of Science][Medline]
  19. Szymanska G, Stromer H, Kim DH, Lorell BH, Morgan JP. Dynamic changes in sarcoplasmic reticulum function in cardiac hypertrophy and failure. Pflügers Arch 439: 339–348, 2000.[CrossRef][Web of Science][Medline]



This article has been cited by other articles:


Home page
 Circ. Res.Home page
P. Nicolaou, P. Rodriguez, X. Ren, X. Zhou, J. Qian, S. Sadayappan, B. Mitton, A. Pathak, J. Robbins, R. J. Hajjar, et al.
Inducible Expression of Active Protein Phosphatase-1 Inhibitor-1 Enhances Basal Cardiac Function and Protects Against Ischemia/Reperfusion Injury
Circ. Res., April 24, 2009; 104(8): 1012 - 1020.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
A. El-Armouche, K. Wittkopper, F. Degenhardt, F. Weinberger, M. Didie, I. Melnychenko, M. Grimm, M. Peeck, W. H. Zimmermann, B. Unsold, et al.
Phosphatase inhibitor-1-deficient mice are protected from catecholamine-induced arrhythmias and myocardial hypertrophy
Cardiovasc Res, December 1, 2008; 80(3): 396 - 406.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
J. Davis, M. V. Westfall, D. Townsend, M. Blankinship, T. J. Herron, G. Guerrero-Serna, W. Wang, E. Devaney, and J. M. Metzger
Designing Heart Performance by Gene Transfer
Physiol Rev, October 1, 2008; 88(4): 1567 - 1651.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
N. Bruchert, N. Mavila, P. Boknik, H. A. Baba, L. Fabritz, U. Gergs, U. Kirchhefer, P. Kirchhof, M. Matus, W. Schmitz, et al.
Inhibitor-2 prevents protein phosphatase 1-induced cardiac hypertrophy and mortality
Am J Physiol Heart Circ Physiol, October 1, 2008; 295(4): H1539 - H1546.
[Abstract] [Full Text] [PDF]

Home page
 PhysiologyHome page
Y. Ikeda, M. Hoshijima, and K. R. Chien
Toward Biologically Targeted Therapy of Calcium Cycling Defects in Heart Failure
Physiology, February 1, 2008; 23(1): 6 - 16.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/1/H762    most recent
00104.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (10)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rodriguez, P.
Right arrow Articles by Kranias, E. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rodriguez, P.
Right arrow Articles by Kranias, E. G.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.