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Cardiac SR-coupled PP1 activity and expression are increased and inhibitor 1 protein expression is decreased in failing hearts

Division of Cardiovascular Medicine, Department of Medicine, Henry Ford Heart and Vascular Institute, Detroit, Michigan 48202

Submitted 19 May 2003 ; accepted in final form 15 July 2003

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Type 1 protein phosphatase (PP1) is a negative regulator of cardiac function. However, studies on the status and regulation of sarcoplasmic reticulum (SR)-associated PP1 activity in failing hearts are limited. We studied PP1 activity and protein and mRNA expression of the catalytic subunit of PP1 (PP1C) and protein levels of PP1-specific inhibitors [inhibitor 1 (Inh-1) and inhibitor 2 (Inh-2)] in the left ventricular (LV) myocardium of 6 dogs with heart failure (HF; LV ejection fraction, 23 ± 2%) and 6 normal dogs. In failing LV tissue, PP1 activity values (expressed as pmol ³²P · min^–1 · mg of noncollagen protein^–1) in the homogenate, crude membranes, cytosol, and purified SR were increased by 52, 54, 55, and 72%, respectively. Trypsin treatment released PP1 but not type 2A protein phosphatase from the SR. In the supernatant of trypsin-treated SR, PP1 activity was 24% higher in failing hearts than in normal control hearts. A similar increase in protein expression of PP1C was observed in the nontrypsinized SR. Heat-denatured phosphorylated SR inhibited PP1 activity by 30%, which suggests the presence of Inh-1 or -2 or both in the SR. With the use of a specific antibody, both Inh-1 and -2 proteins were found in the SR; the former was decreased by 56% in the failing SR, whereas the latter did not change. These results suggest that protein phosphatase activity bound to the SR is increased and is predominantly type 1. Increased SR-associated PP1 activity in failing hearts appears to be due partly to increased expression of PP1C and partly to reduced levels of Inh-1 but not Inh-2 protein. Thus inhibition of PP1 activity in the SR appears to be a potential therapeutic target for improving LV function in failing hearts, because it may lead to increased SR Ca²⁺ uptake, which is impaired in failing hearts.

heart failure; sarcoplasmic reticulum; type 1 protein phosphatase

Although Tyr, Ser/Thr, and dual-specificity PPs are involved in the regulation of hypertrophy, apoptosis, and contractility (15, 17), studies on different subtypes of Ser/Thr PP in failing hearts are limited. Calcineurin, also known as Ca²⁺-calmodulin-dependent PP, type 2B PP (PP2B), or heat-labile inhibitor of Ca²⁺-calmodulin-dependent phosphodiesterase, has been shown to induce cardiac hypertrophy (28), whereas type 2A PP (PP2A) is suggested to be a substrate for caspase-3 during apoptosis (36), and type 1 PP (PP1) may be involved in regulating cardiac contractility (3, 5, 24, 31). In explanted failed hearts with dilated cardiomyopathy, PP activity was found to be increased (25, 30). In one recent study (5), overexpression of the catalytic subunit of PP1C in transgenic mouse hearts caused hypertrophy and dilation. Moreover, the left ventricular (LV) ejection fraction was reduced as was the phosphorylation of PLB, which is a regulator of cardiac sarcoplasmic reticulum (SR) Ca²⁺-ATPase activity. We and others have reported that PLB is present in a dephosphorylated form at Ser16 in the LV myocardium of human and experimental failed hearts (20, 25, 27, 35). This reduced PLB phosphorylation is due to increased PP1 activity rather than changes in cAMP-dependent protein kinase activity (22). Although PP1 activity is increased in failing hearts, it is not clear whether this increased activity is associated with the SR, where PLB is located, or how SR-associated PP1 is regulated in failing hearts.

We have reported that PP activity is increased in the LV myocardium of dogs with HF produced by intracoronary microembolizations (9, 14). In the present study, we found that increased PP activity in failing hearts was associated with the SR and was predominantly of the subtype PP1. The increase in the latter enzyme was partly associated with increased expression of PP1C and partly due to reduced protein expression of inhibitor 1 (Inh-1) but not inhibitor 2 (Inh-2). Increased PP1 activity in the SR can account for reduced SR Ca²⁺ uptake and therefore reduced LV function, which is a characteristic of HF. Thus drugs that inhibit SR-associated PP1 activity may help cure or at least retard the development of LV dysfunction during progression of HF.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Model preparations. Twelve dogs of either sex (19–25 kg body wt) were studied. HF was produced in six dogs, and the other six served as controls. To produce chronic HF, dogs underwent a series of cardiac catheterizations and intracoronary microembolizations as described previously (34). The microembolizations were performed 1–3 wk apart and were discontinued when the LV ejection fraction, as determined angiographically, was close to 35%. Dogs with HF were euthanized 4 mo after the last embolization. Hemodynamic and angiographic measurements were made at baseline and 4 mo after the last embolization, just before the dog was euthanized (33, 34). The study was approved by the Henry Ford Hospital Institutional Animal Care and Use Committee and conformed to the "Position of the American Heart Association on Research Animal Use" and "Guiding Principles in the Care and Use of Animals" of the American Physiological Society. On the last day, after all measurements were completed, the chest was opened through a left thoracotomy, the pericardium was exposed, and the heart was quickly removed and immersed in ice-cold cardioplegic solution. LV myocardial tissue (5 g) from each dog was cut into 5-mm³ blocks (free of obvious scars or epicardial vessels), quickly frozen in liquid nitrogen, and pulverized using a mortar and pestle. LV powder (1 g) was immersed in 10 ml of RNAlater to isolate RNA, and the remainder was stored at –70°C for biochemical, immunoblot, and molecular biological analysis.

Preparation of homogenate, crude membranes, cytosol, and purified SR. Frozen LV tissue (2 g) from each dog was used to prepare the subcellular fractions, which were carried out at 4°C. Specimens were thawed in 10 ml of homogenization buffer that consisted of 50 mM Tris · HCl (pH 7.4), 0.5 mM sodium EDTA (pH 7.0), 0.3 M sucrose, and protease inhibitors (0.8 mM benzamidine, 0.8 mg/l each aprotinin and leupeptin, and 0.4 µg/l antipain). Thawed tissue was homogenized as described previously (10, 13), and the resulting homogenate was filtered through four layers of cheesecloth. About 0.5 ml of Filtrate (homogenate) was saved, and the remainder was centrifuged at 100,000 g for 40 min. We saved 1 ml of clear supernatant (cytosol) and discarded the rest. The pellet was resuspended in 100 ml of homogenization buffer that contained 0.6 M KCl and was centrifuged at 100,000 g for 40 min. The resulting pellet was resuspended in 30 ml of homogenization buffer, centrifuged again, and then resuspended in 2 ml of medium 1 [20 mM Tris · HCl (pH 7.4) and 0.3 M sucrose] to represent a crude membrane. A 0.5-ml aliquot of crude membranes was saved, and the remainder was processed for purification of the SR as described previously (8). The purified SR and crude membranes were assayed for oxalate-dependent SR Ca²⁺ uptake at 10 µM free Ca²⁺ as described previously (10). Ca⁺ uptake measurements in the purified SR and crude membranes (expressed as nmol ⁴⁵Ca²⁺ accumulated · mg protein^–1 · 10 min^–1) were 1,208 ± 79 and 230 ± 18, respectively, which suggests that the SR was purified 5.2-fold. The yield of purified SR (1.5 ± 0.05 mg/g of LV powder) was similar between dogs with HF and control animals. We have reported that oxalate-dependent SR Ca²⁺ uptake and Ca²⁺-ATPase activity and expression are significantly reduced in the LV myocardium in the same canine model of HF (10, 13). All fractions were aliquoted into 100-µl portions, immediately frozen in liquid nitrogen, and stored at –70°C until use.

PP1 activity assay. With the use of [³²P]phosphorylase a as the substrate, PP activity was determined in the homogenate, crude membranes, cytosol, and purified SR fractions in the absence and presence of 600 ng of rabbit skeletal muscle Inh-2. The assay was performed in a 50-µl aliquot that consisted of 50 mM Tris · HCl (pH 7.4), 5 mM caffeine, 0.5 mM EGTA, 0.5 mM EDTA, 50 µM -mercaptoethanol, and 100 ng of aprotinin (protease inhibitor) with or without 600 ng of rabbit skeletal muscle purified Inh-2, the cell fraction (in µg: 2 of homogenate or cytosol, 10 of crude membranes, and 5 of purified SR), and 550 pmol [³²P]phosphorylase a. The assay was initiated by adding the cell fraction and was incubated at 35°C for 5 min. Incubation was rapidly stopped by addition of 30 µl of 60% TCA and 20 µl of BSA (50 mg/ml). Tubes were held in ice for 10 min and then centrifuged at 12,000 g for 5 min. After centrifugation, ³²P radioactivity was counted in 80 µl of clear supernatant in 7 ml of liquid scintillation fluid. PP1 activity was calculated by determining Inh-2-sensitive PP activity, which we obtained by deducting phosphatase activity assayed in the presence of Inh-2 from that assayed in its absence. Activity in each fraction (expressed as pmol ³²P released · min^–1 · mg^–1 of noncollagen protein) was determined by Lowry's method (23).

Trypsin treatment, phosphorylation, and heat denaturation of SR. Purified SR (100 µg) from each dog was treated with trypsin (0–0.4 mg/ml) in a 100-µl aliquot at 34°C for 5 min. The effect of the trypsin was neutralized by adding threefold excess trypsin inhibitor, and the resulting mixture was centrifuged at 100,000 g for 30 min. The supernatant was saved, and the pellet was resuspended in 1 ml of buffer A [50 mM Tris · HCl (pH 7.4), 0.5 mM EGTA, 0.3 M sucrose, and 1 mg/ml trypsin inhibitor] and recentrifuged at the same speed for 30 min. The resulting pellet was resuspended in 100 µl of 50 mM Tris · HCl (pH 7.4) that contained 0.3 M sucrose and 0.5 mg/ml trypsin inhibitor and was stored at 4°C. In both the supernatant and the pellet suspension, PP1 activity was determined as described above. For SR phosphorylation and heat denaturation of the purified SR, 100 µg of SR was phosphorylated by the bovine heart purified catalytic subunit of cAMP-dependent protein kinase as described for Inh-1 (30). After phosphorylation, the assay mixture was centrifuged at 100,000 g for 30 min and the resulting pellet was resuspended in 100 µl of buffer A without trypsin inhibitor and then immersed in a boiling-water bath for 20 min. Heat-treated samples were homogenized gently with a mortar and pestle to obtain an even SR suspension. The effect of the heat-treated SR on PP1 activity was determined as described above, except that the rabbit skeletal muscle purified catalytic subunit of PP1 was added in place of the cell fraction.

Quantitation of mRNA by RT-PCR. Total cellular RNA was isolated from frozen LV tissue in RNAlater solution (Ambion) using RNA STAT-60 according to the manufactur-er's instructions (Tel-Test B; Friendswood, TX). Concentration and quality of the isolated RNA in each sample were determined spectrophotometrically, considering an absorbance ratio of 260:280 nm between 1.7 and 2.0 as good quality. In addition, the isolated RNA exhibited three major bands (28S, 18S, and 5.8S) on 2% agarose with 28S being much stronger than 18S. RNA (8 µg) was reverse-transcribed into cDNA in an 80-µl assay volume. The assay contained (final concentration) 3.6 mM of each dNTP (dATP, dTTP, dGTP, and dCTP), 40 U of recombinant RNasin (RNase inhibitor, Promega), 6 µM oligo(dT) primer, and 1 U of Moloney murine leukemia virus reverse transcriptase in 10 mM Tris · HCl (pH 8.3), 75 mM KCl, 10 mM DTT, and 3.0 mM MgCl₂. Assay tubes were incubated at 42°C for 60 min and then at 96°C for 10 min for denaturation. For each PCR reaction, 2 µl of first-strand cDNA was added to 18 µl of a reaction mixture that contained 20 pmol of each phosphatase forward and reverse primer, 200 µM of each dNTP, 10 mM Tris · HCl (pH 8.8), 50 mM KCl, 0.1% Triton X-100, and 3.0 mM MgCl₂. After the tube was heated to 95°C for 5 min, 1 U of platinum Taq DNA polymerase (Invitrogen; Carlsbad, CA) was added, and PCR was allowed to proceed for 20–40 cycles. PCR products were analyzed by subjecting 10 µl of each reaction mixture to electrophoresis on 1.5% ethidium-bromide-agarose gels. Band sizes of the products were compared with standard DNA size markers and confirmed by sequencing (Wayne State University). The forward and reverse primers for PP1C (forward, 5'-GCCATGTCCGACAGCGAGAAG-3'; reverse, 5'-TCCATGTTCCCCGTGACAGGTG-3') and GAPDH (forward, 5'-ACCACCATGGAGAAGGCTGG-3'; reverse, 5'-CTCAGTG TAGCC CAGG AT GC-3') were based on the gene sequences we reported to GenBank (26). The amplified products exhibited 1.12-and 0.528-kb product sizes for PP1C and GAPDH, respectively. Band intensity (expressed as optical density x mm²) was quantified using a Bio-Rad (San Francisco, CA) GS-670 imaging densitometer.

Western blotting. To determine tissue levels of PP1C and Inh-1 and -2, Western blotting was performed on SDS extracts of the purified SR as described previously for SR proteins in crude membrane fractions (25, 27). Calsequestrin (CSQ), a Ca²⁺-binding protein located in the SR and reported to be unchanged in failing hearts (13), was also quantitated in each sample to normalize protein loading on the gel. Equal volumes of the SDS extract and sample buffer [62.5 mM Tris · HCl (pH 6.8), 20% glycerol, 40 mM DTT, and 0.001% bromophenol blue] were combined, and the resulting mixture was incubated in boiling water for 10 min. An aliquot of the boiled mixture was subjected to electrophoresis on 4–20% SDS-polyacrylamide gel (Bio-Rad), and the separated proteins were electrophoretically transferred to a nitrocellulose membrane (10, 13). The accuracy of the electrotransfer was confirmed by staining the membrane with 0.1% amido black. For the immunoreaction, the nitrocellulose blot was incubated with diluted primary antibody (monoclonal or polyclonal) based on the supplier's instructions. Antibody binding protein(s) was visualized by autoradiography after treating the blot with horseradish peroxidase-conjugated secondary antibody (anti-rabbit) and enhanced chemiluminescence color-developing reagents according to the supplier (Amersham). The intensity of the band was quantified using a Bio-Rad GS-670 imaging densitometer (expressed as densitometric units x mm²). In all circumstances, we made sure the antibody was present in excess over the antigen and the density of each protein band was in the linear scale.

Miscellaneous methods. Inh-2 was purified from rabbit skeletal muscle as described previously (38). The protein obtained after Sepharose CL-6B column chromatography was free of Inh-1 and was 50–60% pure. Its yield was 3 mg/kg of fresh rabbit skeletal muscle. Inh-1 and -2 in dog LV homogenate were separated by DEAE cellulose chromatography, and each inhibitor peak was processed separately for purification involving ammonium sulfate precipitation (25–55%), heat denaturation, and Sepharose CL-6B chromatography. Inh-1 or -2 obtained at this step was purified further using a Superose-12 gel filtration column. The amount of purified protein was 0.5 mg for Inh-1 and 0.2 mg for Inh-2 per kilogram of dog LV tissue. PP1C was purified from rabbit skeletal muscle as described previously (37). Briefly, fresh rabbit skeletal muscle was homogenized and centrifuged. The resulting clear supernatant was treated with ethanol to precipitate the catalytic subunits of PP1 and PP2A, which were separated by DEAE-cellulose column chromatography. The activity peak corresponding to PP1 and PP2A was pooled separately, concentrated, dialyzed, and centrifuged, and the resulting solution was stored at –80°C. Rabbit skeletal muscle purified PP1C was 50% pure as assessed by SDS-polyacrylamide gel electrophoresis. The bovine heart catalytic subunit of cAMP-dependent protein kinase was purified as described previously (11). A polyclonal antibody was raised against the rabbit skeletal muscle purified Inh-1 of PP1 according to the standard protocol as described previously (16). The antibody interacted with Inh-1 but not Inh-2 (data not shown).

Materials. Antibodies specific for PP1C and Inh-2 were purchased from Transduction Laboratories, and an antibody specific for CSQ was obtained from Dr. Larry R. Jones (Krannert Institute of Cardiology; Indianapolis, IN). Chemicals and supplies for electrophoresis and electrotransfer were purchased from Bio-Rad (San Francisco, CA). Biochemical supplies were obtained from Sigma Chemical (St. Louis, MO). Primers of the different genes were synthesized by Invitrogen, and RT-PCR products were sequenced by the DNA Sequencing facility at Wayne State University (Detroit, MI).

Statistical analysis. Data are means ± SE. Comparisons between failing and nonfailing hearts were based on Student's t-test for two means (unpaired t-test), considering P < 0.05 as significant. The sample size used in this study of 6 failing and 6 nonfailing hearts was based on 80% power to detect a large difference at = 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Hemodynamic and angiographic data in dogs with HF. Table 1 shows the hemodynamic and angiographic measurements obtained at baseline and 4 mo after the last embolization in dogs with HF. Aortic pressure and heart rate values were measured in all dogs. The heart rate changed significantly, whereas aortic pressure did not change in dogs with HF (Table 1). LV ejection fraction, LV peak isovolumic contraction and relaxation (+dP/dt and -dP/dt), and stroke volume values were all significantly decreased in HF dogs and were associated with significant increases in LV end-diastolic volume (70 ± 7 vs. 54 ± 6 ml; P < 0.05) and pressure (23 ± 3 vs. 6 ± 1 mmHg; P < 0.05). These results are consistent with LV failure. In our laboratory, we have used the same canine model of microembolization-induced HF in several studies, and results were always reproducible (10, 13, 33, 34).

PP activity. Cardiac tissue contains several subtypes of Ser/Thr PP including PP1 and PP2A (15). To determine PP1 activity, we measured the amount of rabbit skeletal muscle purified Inh-2 (a potent inhibitor of PP1) that was needed to completely inhibit the PP1 activity associated with the cytosol and membranes isolated from LV tissue and found that it took 320 ng for cytosol and 500 ng for membranes (Fig. 1). In this assay, we used 50% purified Inh-2 and PP1C. On the basis of a molecular mass of 32 kDa for Inh-2 and assuming 1 mol of Inh-2 would inhibit 1 mol of PP1C having a molecular mass of 38 kDa, the percentage of PP1C was 9.5% in the cytosol and 2.97% in the membrane of the LV myocardium of normal dogs. PP1 activity levels in the homogenate, cytosol, membranes, and purified LV SR are shown in Fig. 2. PP1 activity (expressed as pmol ³²P · min^–1 · mg of noncollagen protein^–1) increased significantly in the homogenate by 52% (1,868.50 ± 50 vs. 1,226.30 ± 49; n = 6; P < 0.05), 54% in the crude membranes (458 ± 13 vs. 298 ± 30; n = 6; P < 0.05), and 55% in the cytosol (1,531.20 ± 110 vs. 990 ± 56; n = 6; P < 0.05) as indicated in Fig. 2A, whereas PP1 in the purified SR increased by 73% (2,386 ± 100 vs. 1,377 ± 53; n = 6; P < 0.05) as shown in Fig. 2B. However, there was no difference in PP2A activity in purified SR between dogs with HF and control animals (273.2 ± 11 vs. 272.5 ± 8 pmol ³²P · min^–1 · mg of protein^–1; n = 6; P < 0.55; Fig. 2B). When PP2A activity (expressed as pmol ³²P · min^–1 · mg of noncollagen protein^–1) was measured in different fractions of LV tissue, it increased by 27% in the homogenate (1,090.00 ± 39 vs. 865.33 ± 16; P < 0.05) and 39% in the cytosol (1,242.17 ± 25 vs. 890.83 ± 29; P < 0.05) but did not change significantly in crude membranes of the LV myocardium of dogs with HF compared with normal dogs. Because the failing heart undergoes hypertrophy and fibrosis (34), we determined SR-associated PP1 activity in the whole LV myocardium in addition to activity per milligram of noncollagen protein. Noncollagen protein (0.86 ± 0.02 vs. 0.78 ± 0.01 mg/mg of LV tissue) and LV wt normalized to body wt (4.37 ± 0.07 vs. 4.22 ± 0.08 g/kg) were slightly higher in dogs with HF, but this was not statistically significant. The amount of purified SR obtained from scarfree LV tissue of dogs with HF was not significantly different from control dogs (1.85 ± 0.2 vs. 1.90 ± 0.2 mg/g of LV tissue; n = 6). Based on these values, total PP1 activity associated with purified SR in the whole LV myocardium of dogs with HF was even higher: 75% compared with control dogs (19,278.9 ± 1,156 vs. 11,043.5 ± 607 pmol ³²P/min per whole LV myocardium; n = 6; P < 0.05).

View larger version (17K): [in this window] [in a new window]   Fig. 1. Effects of type 1 protein phosphatase (PP1)-specific inhibitor 2 (Inh-2) on protein phosphatase (PP) activity in the cytosol and membranes isolated from the left ventricular (LV) myocardium of normal dogs and rabbit skeletal muscle purified catalytic subunit of PP1 (PP1C). Rabbit skeletal muscle purified PP1C was 0.6 µg. PP activity was determined using [32P]phosphorylase a as substrate. Values are means ± SE of 3 different experiments.

View larger version (24K): [in this window] [in a new window]   Fig. 2. A: PP1 activity in the homogenate, membrane, and cytosol isolated from the LV myocardium of normal (NL) dogs and dogs with heart failure (HF). B: PP1 and type 2A PP (PP2A) activities in purified sarcoplasmic reticulum (SR) isolated from the LV myocardium. Homogenate, membrane, cytosol, and purified SR were prepared from the left ventricle. In these isolated fractions, PP1 and PP2A activities were determined using [32P]phosphorylase a as substrate. Values are means ± SE of 6 NL dogs and 6 dogs with HF; *P < 0.05 vs. NL was considered significant.

PP1C expression levels. To examine whether increased phosphatase activity in failing hearts is associated with increased expression of PP1C, protein expression was measured by two methods, trypsin analysis and Western blotting, and mRNA expression was measured by RT-PCR. After treatment of the SR with varying trypsin concentrations (0–0.4 mg/ml), the suspension was centrifuged and PP1 activity was determined in the pellet and supernatant. PP1 activity declined in the pellet but increased in the supernatant, which suggests release of PP1C from the SR (Fig. 3A). SRs of control animals and dogs with HF were treated with 0.1 mg/ml of trypsin, followed by centrifugation, and PP1 and PP2A activities were determined in the pellets and supernatants. Trypsin (0.1 mg/ml) released 70% activity in the supernatant (Fig. 3B), whereas 23% activity remained bound to the pellet (Fig. 3C). When PP1 activity was measured in the trypsin-solubilized supernatant (0.1 mg/ml), it was increased by 24% in the supernatant of dogs with HF compared with control dogs (1,612 ± 88 vs. 1,296 ± 42; Fig. 3B) in contrast to a 55% increase in the pellet (219 ± 12 vs. 141 ± 10; Fig. 3C), which suggests that PP1 activity is regulated differently depending on whether it is bound to the SR. Interestingly, trypsin did not dissociate PP2A activity from the SR (Fig. 3C). There was no difference in PP2A activity in the trypsin-treated SR between control dogs and dogs with HF (Fig. 3B).

View larger version (20K): [in this window] [in a new window]   Fig. 3. A: assay of PP activity in the supernatant and pellet of the SR after treatment with 0–0.4 mg/ml trypsin. B: assay of PP1 and PP2A in the supernatant of trypsin-treated (0.1 mg/ml) SR isolated from the LV myocardium of NL dogs and dogs with HF. C: assay of PP1 and PP2A in the pellet of trypsin-treated (0.1 mg/ml) SR isolated from the LV myocardium of NL and HF dogs. SR reaction mixture was centrifuged to collect the supernatant and pellet, and phosphatase activity was determined in both fractions. Values are means ± SE of 6 NL dogs and 6 dogs with HF; *P < 0.05 vs. NL was considered significant.

We determined mRNA levels of PP1C in total cellular RNA (Fig. 4). Before quantitating mRNA, we optimized PCR cycle numbers to ensure formation of PCR products in the linear range for PP1C and GAPDH, which is a housekeeping gene that remains unchanged between control animals and dogs with HF (Fig. 4A; Ref. 27). Expression of PP1C mRNA (1.07 kb) but not GAPDH (0.542 kb) was higher in dogs with HF (Fig. 4B). Densitometric analysis of PP1C normalized to GAPDH exhibited an 80% increase in dogs with HF compared with control dogs (0.72 ± 0.05 vs. 0.4 ± 0.03; n = 6; P < 0.05). These results suggest higher mRNA expression than protein expression of PP1C associated with the purified SR. With the use of an antibody that recognizes all isoforms of PP1C, protein expression of PP1C was determined in the purified SR from controls and dogs with HF (Fig. 5). CSQ expression was also determined to normalize protein loading, because CSQ is not altered in failing hearts (13). Both antibodies detected a single band in varying concentrations of purified SR that measured 38 kDa for PP1C and 55 kDa for CSQ (Fig. 5A). When PP1C and CSQ were quantitated in 20 µg of purified SR from the LV myocardium, PP1C increased, whereas CSQ remained unchanged in dogs with HF compared with control dogs (Fig. 5B). Densitometric analysis of the PP1C normalized to CSQ showed an 37% increase in PP1C in dogs with HF (0.44 ± 0.03 vs. 0.32 ± 0.02; n = 6; P < 0.05; Fig. 5C).

View larger version (40K): [in this window] [in a new window]   Fig. 4. A: effects of varying PCR cycle numbers on mRNA expression of the -isoform of the catalytic subunit of PP1 (PP1C) and GAPDH in total RNA isolated from LV tissue of NL dogs. B: mRNA expression of PP1C and GAPDH in LV tissue of 2 NL and 2 HF dogs. C: densitometric analysis of PP1C normalized to GAPDH in LV tissue of 6 NL and 6 HF dogs; *P < 0.05 vs. NL was considered significant.

View larger version (24K): [in this window] [in a new window]   Fig. 5. A: Western blot shows immunodetectable levels of PP1C and calsequestrin (CSQ) in varying concentrations of the SR (5–80 µg) isolated from LV tissue of NL dogs. B: Western blot shows immunodetectable levels of PP1C and CSQ in SR (20 µg) isolated from LV tissue of 2 NL dogs and 2 dogs with HF. C: densitometric analysis of PP1C normalized to CSQ in LV tissue of 6 NL and 6 HF dogs; *P < 0.05 vs. NL was considered significant.

PP1 inhibitors. The divergent increase in PP1 activity associated with the SR before and after trypsin treatment suggested different regulation of the enzyme. PP1 activity is regulated by Inh-1 and -2, which are the two most potent PP1 inhibitors (17); however, their presence in dog cardiac tissue has not been documented to our knowledge. Because of the thermal stability of these inhibitors, the purified SR was heat treated before examination of its effects on PP1 activity. Heat-treated SR inhibited PP1 activity by 30% in a concentration-dependent manner (Fig. 6A), which suggests the presence of inhibitor-like proteins in the SR. The higher increase in PP1 activity in the failing SR could be partly due to dephosphorylation of Inh-1 caused by activation of calcineurin, an enzyme reported to primarily dephosphorylate Inh-1 out of all those substrates tested (19) and therefore likely to cause a higher increase in PP1 activity. If so, phosphorylation of the SR by cAMP-dependent protein kinase would partly reduce the percent increase in PP1 activity in the failing SR compared with controls. We measured PP1 and PP2A activity after phosphorylation of the SR by cAMP-dependent protein kinase. PP2A activity associated with the purified SR was not affected. However, the increase in PP1 activity in the failing vs. nonfailing SR was only 25% (Fig. 6B) compared with 75% before phosphorylation (see Fig. 2B). With the use of the Inh-2 antibody, a 32-kDa protein was recognized in the purified SR (Fig. 7A); however, there was no difference in Inh-2 protein expression (Fig. 7B). Densitometric analysis of Inh-2 normalized to CSQ also showed no difference (Fig. 7C). With the use of an antibody, Inh-1 was detected as a 26-kDa protein in the purified SR. When Inh-1 levels were quantitated in 120 µg of purified SR, it was decreased in dogs with HF compared with normal dogs (Fig. 8A). Densitometric analysis of Inh-1 normalized to CSQ showed an 56% decrease in the purified SR of dogs with HF (0.21 ± 0.02 vs. 0.48 ± 0.02; Fig. 8B).

View larger version (19K): [in this window] [in a new window]   Fig. 6. A: effects of varying concentrations of heat-denatured phosphorylated SR from NL LV tissue on activity of the rabbit skeletal muscle purified catalytic subunit of PP1. Purified SR was phosphorylated by cAMP-dependent protein kinase and then heat denatured. Phosphatase activity in the absence of phosphorylated heat-denatured SR was considered 100%. B: effects of cAMP-dependent protein kinase-induced phosphorylation on PP1 and PP2A activity associated with purified SR of 6 NL and 6 HF dogs. SR (50 µg) was phosphorylated and heat denatured, and the effects of the treated SR on activity of rabbit skeletal muscle purified PP1C (0.6 µg) were examined. *P < 0.05 vs. NL was considered significant.

View larger version (40K): [in this window] [in a new window]   Fig. 7. A: Western blot shows immunodetectable Inh-2 in varying concentrations of purified SR of LV tissue from a NL dog. B: Western blot shows immunodetectable Inh-2 in purified SR of LV tissue from 2 NL and 2 HF dogs. C: densitometric analysis of immunodetectable Inh-2 normalized to CSQ in 6 NL and 6 HF dogs. Inh-2 purified from the cytosol of dog LV tissue (DLV Inh-2) was used as a control.

View larger version (33K): [in this window] [in a new window]   Fig. 8. A: Western blot shows immunodetectable Inh-1 in purified SR of LV tissue from 2 NL and 2 HF dogs. B: densitometric analysis of immunodetectable Inh-1 normalized to CSQ in 6 NL and 6 HF dogs. *P < 0.05 vs. NL was considered significant. Inh-1 purified from the cytosol of dog LV tissue (DLV Inh-1) was used as a control.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
The present results demonstrate that PP activity is increased in the LV myocardium of dogs with HF. The increase in enzyme activity is associated with the isolated SR and is due to an alteration in PP1 but not PP2A. Moreover, this increased enzyme activity is due partly to increased enzyme expression and partly to reduced Inh-1 protein in the SR. PP1C enzyme is bound to the SR through an anchoring protein that releases PP1C upon trypsinization but not phosphorylation with protein kinase activity similar to glycogen-binding PP1C of rabbit skeletal muscle (17, 19). These findings strengthen our previous observations of reduced phosphorylation of PLB in the LV myocardium of dogs with HF (27). Thus increased PP1 activity in the SR may lead to reduced phosphorylation of PLB (an intrinsic protein of the SR) at Ser16 and Thr17. Furthermore, this increase in PP1 activity may be partly responsible for reduced SR Ca²⁺ uptake, which is a characteristic of HF that has been recognized by many investigators.

Reversible protein phosphorylation is an important signaling mechanism that leads to cardiac hypertrophy and HF. The former is controlled by a balance between protein kinases and PPs, which are regulated by intracellular second messengers generated in response to external stimuli (17). Unlike protein kinases, very little is known about regulation of PPs in normal and diseased hearts. For the last decade, regulation of calcineurin as a stimulus for cardiac hypertrophy and HF has been understood in detail (15, 17, 28); however, regulation of other subtypes of Ser/Thr PPs such as PP1, PP2A, and PP2C remains poorly understood. In one recent study (5), PP1C overexpression in the transgenic mouse heart led to the development of LV hypertrophy and HF. In experimental and explanted human failed hearts, several studies reported increased PP1 activity, expression, or both (5, 9, 14, 20, 25, 30, 35). In the present study, we clearly demonstrated that PP1 activity was increased significantly by 52% in the homogenate, 54% in the crude membranes, and 55% in the cytosol, whereas PP1 in the purified SR was increased by 73% in the LV myocardium of dogs with HF compared with normal dogs. Purified SR from normal LV myocardium contains 17% PP2A and 83% PP1 subtypes. However, PP activity in the failing LV SR involves an increase in PP1 but not PP2A, which is similar to experimental and human failing hearts (20, 30). In both studies, although the purified SR was not studied, similar results were observed in crude membrane fractions, which suggests that the SR is the principal membrane fraction for increased PP1 activity in failing hearts. Increased SR-associated PP1 activity in failing hearts is in good agreement with reports of reduced PLB phosphorylation at Ser16 and Thr17 in human or experimental failed hearts (20, 25, 35). Reduced PLB phosphorylation in the failing SR could also be partly due to reduced protein kinase activity and Ca²⁺-calmodulin-dependent protein kinase. The former remains unchanged in failing hearts, whereas the status of the latter is controversial (22, 27, 29). These results suggest that increased PP1 activity may be partly responsible for reduced SR Ca²⁺ uptake in failing hearts through reduced PLB phosphorylation. Because phosphorylation of PLB modulates SR Ca²⁺-ATPase activity and therefore cardiac contraction, inhibition of PP1 activity is a potential target for development of drugs that can increase LV function by improving PLB phosphorylation in the SR.

Although some laboratories reported increased PP activity in failed hearts, few (30, 35) studied the molecular mechanisms behind the increases in PP1 activity. One possible mechanism may be an increase in transcriptional and translational levels of the PP1C. Both mRNA and protein levels were found to be elevated in the LV myocardium of dogs with HF. In the present study, we observed an 24% increase in the protein level of SR-associated PP1C in failing hearts, which is similar to rats with HF produced by ligation of the left anterior descending coronary artery (35). In addition to protein, we also observed an 80% increase in PP1C mRNA in failing hearts, which is similar to explanted failed human hearts (30). However, the latter did not include protein levels of PP1C. Because we observed an 73% increase in SR-associated PP1C activity but only a 37% increase in PP1C protein, these findings suggest that PP1C bound to the SR might be regulated by some other mechanism(s).

Another mechanism for the increase in PP1 activity could be changes in PP1 inhibitory proteins Inh-1 and -2, which have long been recognized in skeletal muscle (17); however, in cardiac tissue, Inh-1 has been studied only superficially in relation to regulation of PP1 activity and Inh-2 has not been investigated at all. Studies have shown that Inh-1 is active only when it has been phosphorylated by cAMP-dependent protein kinase. In one recent study, Inh-1 was found to exist in a dephosphorylated form in cardiomyocytes isolated from explanted failed human LV myocardium with idiopathic dilated cardiomyopathy (5). We also reported that reduced phosphorylation but not protein expression of PP1 Inh-1 was partly responsible for increased PP activity associated with the membrane of the LV myocardium from dogs with chronic HF (9). Those studies were carried out in LV homogenates instead of purified SR from LV tissue. In the LV homogenate, we (9) and others (5) did not see changes in protein expression between failing and nonfailing hearts. However, in the present study, Inh-1 protein expression was found to be decreased in the purified SR of failing hearts. Reduced PP1 Inh-1 expression may be partly responsible for increased PP1 activity associated with the SR. These findings are in close agreement with the most recent published study (1) in which expression of Inh-1 mRNA decreased in explanted failed human hearts compared with donor nonfailing hearts. Our studies suggest clearly that Inh-1 and -2 are integral proteins that are localized near PP1 in the SR, where they modulate PP1 activity and thereby regulate phosphorylation of PLB, a modulator of SR Ca²⁺-ATPase.

The third possible mechanism for the increase in SR-associated PP1 activity could be dephosphorylation of the protein that anchors PP1C to the SR. In skeletal muscle, earlier studies have suggested that phosphorylation of the anchoring G protein (bound to the glycogen) by cAMP-dependent protein kinase may cause dissociation of PP1C and thereby reduced phosphatase activity of glycogen (19). A similar mechanism was suggested as being operative in the cardiac SR. We tested this hypothesis in microsomal fractions of guinea pig hearts but found that no catalytic subunit of phosphatase was released after phosphorylation by cAMP-dependent protein kinase (12). In the present study, we examined the effects of the heat-denatured phosphorylated SR on phosphatase activity and found a 30% reduction, which suggests that an Inh-1-like protein is present in the SR. It is also possible that reduced PP1C activity by the heat-denatured phosphorylated SR could be partly due to phosphorylation of PLB, which can compete with the phosphatase substrate and thereby reduce phosphatase activity. However, this possibility appears to be highly unlikely because the amount of PLB present in the assay is 20 pmol versus 550 pmol phosphorylase a. Using a specific antibody, we have categorically demonstrated the presence of Inh-1 and -2 in the purified SR. Although phosphorylation of Inh-1 has not been measured because of its very low levels in the SR, our experiments suggest that Inh-1 protein expression is decreased in the SR, and this protein may exist in its dephosphorylated form in failing hearts. Thus reduced expression and phosphorylation may be partly responsible for increased SR-associated PP activity. Increased PP1C activity caused by either an increase in protein level of PP1C or ablation of Inh-1 appears to be responsible for inducing HF as was recently reported for transgenic mouse hearts (5). In the same study, adenoviral overexpression of a constitutively active Inh-1 was associated with salvage of -adrenergic responsiveness in failing human cardiomyocytes. Thus PP1 is an important regulator of cardiac function, and inhibition of its activity may represent a novel therapeutic target for HF.

In summary, we believe this is the first study on large animals to demonstrate that SR-associated PP activity is increased in the LV myocardium of dogs with HF and that this increase involves PP1 but not PP2A. Increased SR-associated PP1 activity is due partly to increased protein expression of PP1C and partly to reduced Inh-1 protein expression and phosphorylation. In addition, cardiac SR-associated PP1 activity does not appear to be regulated by the anchoring protein attached to the SR and PP1C. This increased SR-associated PP1 activity may be responsible for impaired SR function in HF and, hence, LV dysfunction, which is a characteristic feature of failing hearts.

	DISCLOSURES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-49090-09.

	FOOTNOTES

 

Address for reprint requests and other correspondence: R. C. Gupta, Cardiovascular Research, Henry Ford Hospital, 2799 W. Grand Blvd., Detroit, MI 48202 (E-mail: rgupta1{at}hfhs.org).

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 MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 

1. Al-Armouche A, Rau T, Zolk O, Ditz D, Pamminger T, Zimmermann WH, Jackel E, Harding SE, Boknik P, Neumann J, and Eschenhagen T. Evidence for protein phosphatase inhibitor-1 playing an amplifier role in beta-adrenergic signaling in cardiac myocytes. FASEB J 17: 437–439, 2003.[Abstract/Free Full Text]
2. Ayllon V, Cayla X, Garcia A, Roncal F, Fernandez R, Albar JP, Martinez C, and Rebollo A. Bcl-2 targets protein phosphatase 1 alpha to Bad. J Immunol 166: 7345–7352, 2001.[Abstract/Free Full Text]
3. Boknik P, Khorchidi S, Bodor GS, Huke S, Knapp J, Linck B, Luss H, Muller FU, Schmitz W, and Neumann J. Role of protein phosphatases in regulation of cardiac inotropy and relaxation. Am J Physiol Heart Circ Physiol 280: H786–H794, 2001.[Abstract/Free Full Text]
4. Brodde OE, Michel MC, and Zerkowski HR. Signal transduction mechanisms controlling cardiac contractility and their alterations in chronic heart failure. Cardiovasc Res 30: 570–584, 1995.[Web of Science][Medline]
5. Carr AN, Schmidt AG, Suzuki Y, Monte FD, Sato Y, Lanner C, Breeden K, Jing SL, Allen PB, Greengard P, Yatani A, Hoit BD, Grupp IL, Hajjar RJ, DePaoli-Roach AA, and Kranias EG. Type-1 phosphatase, a negative regulator of cardiac function. Mol Cell Biol 22: 4124–4135, 2002.[Abstract/Free Full Text]
6. Chien KR. Stress pathways and heart failure. Cell 98: 555–558, 1999.[Web of Science][Medline]
7. Everett AD, Stoops TD, Nairn AC, and Brautigan D. Angiotensin II regulates phosphorylation of translation elongation factor-2 in cardiac myocytes. Am J Physiol Heart Circ Physiol 281: H161–H167, 2001.[Abstract/Free Full Text]
8. Feher JJ and Davis MD. Isolation of rat cardiac sarcoplasmic reticulum with improved Ca²⁺ uptake and ryanodine binding. J Mol Cell Cardiol 23: 249–258, 1991.[Web of Science][Medline]
9. Gupta RC, Goldstein S, and Sabbah HN. Protein phosphatase activity is augmented and the state of phosphorylation of protein phosphatase inhibitor-1 is lower in myocardium of dogs with chronic heart failure (Abstract). In: New York Academy of Sciences Conference: Cardiac Sarcoplasmic Reticulum Function and Regulation of Cardiac Contractility. Washington, DC, p. PI–17, 1997.
10. Gupta RC, Mishra S, Mishima T, Goldstein S, and Sabbah HN. Reduced sarcoplasmic reticulum Ca²⁺ uptake and expression of phospholamban in left ventricular myocardium of dogs with heart failure. J Mol Cell Cardiol 31: 1381–1389, 1999.[Web of Science][Medline]
11. Gupta RC, Neumann J, Durant P, and Watanabe AM. A₁-adenosine receptor-mediated inhibition of isoproterenol-stimulated protein phosphorylation in ventricular myocytes. Evidence against a cAMP-dependent effect. Circ Res 72: 65–74, 1993.[Abstract/Free Full Text]
12. Gupta RC, Neumann J, Watanabe AM, and Sabbah HN. Inhibition of type 1 protein phosphatase activity by activation of -adrenoceptors in ventricular myocardium. Biochem Pharmacol 63: 1069–1076, 2002.[Web of Science][Medline]
13. Gupta RC, Shimoyama H, Tanimura M, Nair R, Lesch M, and Sabbah HN. SR Ca²⁺-ATPase activity and expression in ventricular myocardium of dogs with heart failure. Am J Physiol Heart Circ Physiol 273: H12–H18, 1997.[Abstract/Free Full Text]
14. Gupta RC, Tanimura M, Mishima T, Lesch M, and Sabbah HN. Protein phosphatase activity is increased and state of phosphorylation of phospholamban is decreased in myocardium of dogs with chronic heart failure (Abstract). In: New York Academy of Sciences Conference: Cardiac Sarcoplasmic Reticulum Function and Regulation of Cardiac Contractility. Washington, DC, p. PI–14, 1997.
15. Hardt SE and Sadoshima J. Glycogen synthase kinase-3b: a novel regulator of cardiac hypertrophy and development. Circ Res 90: 1055–1063, 2002.[Abstract/Free Full Text]
16. Harlow E and Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1988.
17. Herzig S and Neumann J. Effects of serine/threonine protein phosphatases on ion channels in excitable membranes. Physiol Rev 80: 173–210, 2000.[Abstract/Free Full Text]
18. Horiuchi M, Hayashida W, Kambe T, Yamada T, and Dzau VJ. Angiotensin type 2 receptor dephosphorylates Bcl-2 by activating mitogen-activated protein kinase phosphatase-1 and induces apoptosis. J Biol Chem 272: 19022–19026, 1997.[Abstract/Free Full Text]
19. Hubbard MJ and Cohen P. Regulation of protein phosphatase-I_G from rabbit skeletal muscle. 1. Phosphorylation by cAMP-dependent protein kinase at site 2 releases catalytic subunit from the glycogen-bound holoenzyme. Eur J Biochem 186: 701–709, 1989.[Web of Science][Medline]
20. Huang B, Wang S, Qin D, Boutjdir M, and El-Sherif N. Diminished basal phosphorylation level of phospholamban in the postinfarction remodeled rat ventricle: role of -adrenergic pathway, Gi protein, phosphodiesterase, and phosphatases. Circ Res 85: 848–855, 1999.[Abstract/Free Full Text]
21. Kadambi VI and Kranias EG. Genetically engineered mice: model systems for left ventricular failure. J Card Fail 4: 349–361, 1998.[Medline]
22. Kirchhefer U, Schmitz W, Scholz H, and Neumann J. Activity of cAMP-dependent protein kinase and Ca²⁺/calmodulin-dependent protein kinase in failing and nonfailing human hearts. Cardiovasc Res 42: 254–261, 1999.[Abstract/Free Full Text]
23. Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685, 1951.
24. Luss H, Klein-Wiele O, Boknik P, Herzig S, Knapp J, Linck B, Muller FU, Scheld HH, Schmid C, Schmitz W, and Neumann J. Regional expression of protein phosphatase type 1 and type 2A catalytic subunits in the human heart. J Mol Cell Cardiol 32: 2349–2359, 2000.[Web of Science][Medline]
25. Mishra S, Gupta RC, Tiwari N, Sharov VG, and Sabbah HN. Molecular mechanisms of reduced sarcoplasmic reticulum Ca²⁺ uptake in human failing left ventricular myocardium. J Heart Lung Transplant 21: 366–373, 2002.[Web of Science][Medline]
26. Mishra S, Sabbah HN, and Gupta RC. Cloning of catalytic subunit of protein phosphatase type 1 from dog heart. EMBL GenBank DDBJ Database, accession no. AY062037 [GenBank] .1, submitted Nov 2001.
27. Mishra S, Sabbah HN, Jain JC, and Gupta RC. Reduced Ca²⁺-calmodulin-dependent protein kinase activity and expression in LV myocardium of dogs with heart failure. Am J Physiol Heart Circ Physiol 284: H876–H883, 2003.[Abstract/Free Full Text]
28. Molkentin JD, Lu JR, Antos CL, Markham B, Richardson J, Robbins J, Grant SR, and Olson EN. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell 93: 215–228, 1998.[Web of Science][Medline]
29. Nettican T, Temsah RM, Kawabata K, and Dhalla NS. Sarcoplasmic reticulum Ca²⁺/calmodulin-dependent protein kinase is altered in heart failure. Circ Res 86: 596–605, 2000.[Abstract/Free Full Text]
30. Neumann J, Eschenhagen T, Jones LR, Linck B, Schmitz W, Scholz H, and Zimmermann N. Increased expression of cardiac phosphatases in patients with end-stage heart failure. J Mol Cell Cardiol 29: 265–272, 1997.[Web of Science][Medline]
31. Neumann J, Maas R, Boknik P, Jones LR, Zimmermann N, and Scholz H. Pharmacological characterization of protein phosphatase activities in preparations from failing human hearts. J Pharmacol Exp Ther 289: 188–193, 1999.[Abstract/Free Full Text]
32. Roach PJ. Multisite and hierarchal protein phosphorylation. J Biol Chem 266: 14139–14142, 1991.[Abstract/Free Full Text]
33. Sabbah HN, Sharov VG, Lesch ML, and Goldstein S. Progression of heart failure: a role for interstitial fibrosis. Mol Cell Biochem 147: 29–34, 1995.[Web of Science][Medline]
34. Sabbah HN, Stein PD, Kono T, Gheorghiade M, Levine TB, Jafrei S, Hawkins ET, and Goldstein S. A canine model of chronic heart failure produced by multiple sequential intracoronary microembolizations. Am J Physiol Heart Circ Physiol 260: H1379–H1384, 1991.[Abstract/Free Full Text]
35. Sande JB, Sjaastad I, Hoen IB, Bokenes J, Tonnessen T, Holt E, Lunde PK, and Christensen G. Reduced level of serine16 phosphorylated phospholamban in the failing rat myocardium: a major contributor of reduced SERCA2 activity. Cardiovasc Res 53: 382–391, 2002.[Abstract/Free Full Text]
36. Santoro MF, Annand RR, Robertson MM, Peng YW, Brady MJ, Mankovich JA, Hackett MC, Ghayur T, Walter G, Wong WW, and Giegel DA. Regulation of protein phosphatase 2A activity by caspase-3 during apoptosis. J Biol Chem 273: 13119–13128, 1998.[Abstract/Free Full Text]
37. Tung HYL, Resink TJ, Hemmings BA, Sheolikar S, and Cohen P. The catalytic subunits of protein phosphatase-1 and protein phosphatase-2A are distinct gene products. Eur J Biochem 138: 635–641, 1984.[Web of Science][Medline]
38. Yang SD, Vandenheede JR, and Merlevede W. A simplified procedure for the purification of the protein phosphatase modulator (inhibitor-2) from rabbit skeletal muscle. FEBS Lett 132: 293–295, 1981.[Web of Science][Medline]

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