Overexpression of the Catalytic Subunit of Protein Phosphatase 2A Impairs Cardiac Function

Reversible protein phosphorylation is an essential regulatory mechanism in many cellular functions. In contrast to protein kinases, the role and regulation of protein phosphatases has remained ambiguous. To address this issue, we generated transgenic mice that overexpress the catalytic subunit α of protein phosphatase 2A (PP2A) (PP2Acα) in the heart driven by the α-myosin heavy chain promoter. Overexpression of the PP2Acα gene in the heart led to increased levels of the transgene both at RNA and protein levels. This was accompanied by a significant increase of PP2A enzyme activity in the myocardium. Morphological analysis revealed isles of necrosis and fibrosis. The phosphorylation state of phospholamban, troponin inhibitor, and eukaryotic elongation factor 2 was reduced significantly. The expression of junctional (calsequestrin) and free SR proteins (SERCA and phospholamban) was not altered. Whereas no increase in morbidity or mortality was noted, transgenic mice developed cardiac hypertrophy and reduced contractility of the heart, as well as cardiac dilatation as shown by biplane echocardiography. Taken together, these findings are indicative of the fundamental role of PP2A in cardiac function and imply that disturbances in protein phosphatases expression and activity may cause or aggravate the course of cardiac diseases. Reversible protein phosphorylation is an essential regulatory mechanism in many cellular functions. In contrast to protein kinases, the role and regulation of protein phosphatases has remained ambiguous. To address this issue, we generated transgenic mice that overexpress the catalytic subunit α of protein phosphatase 2A (PP2A) (PP2Acα) in the heart driven by the α-myosin heavy chain promoter. Overexpression of the PP2Acα gene in the heart led to increased levels of the transgene both at RNA and protein levels. This was accompanied by a significant increase of PP2A enzyme activity in the myocardium. Morphological analysis revealed isles of necrosis and fibrosis. The phosphorylation state of phospholamban, troponin inhibitor, and eukaryotic elongation factor 2 was reduced significantly. The expression of junctional (calsequestrin) and free SR proteins (SERCA and phospholamban) was not altered. Whereas no increase in morbidity or mortality was noted, transgenic mice developed cardiac hypertrophy and reduced contractility of the heart, as well as cardiac dilatation as shown by biplane echocardiography. Taken together, these findings are indicative of the fundamental role of PP2A in cardiac function and imply that disturbances in protein phosphatases expression and activity may cause or aggravate the course of cardiac diseases. Reversible protein phosphorylation is an essential regulatory mechanism in many cellular functions. Evidence has accumulated that, like protein kinases, protein phosphatases (PP) 1The abbreviations used are: PP, protein phosphatase; eEF-2, eukaryotic elongation factor 2; OA, okadaic acid; PK, protein kinase; PLB, phospholamban; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; SERCA, sarcoplasmic reticulum Ca2+-ATPase; SR, sarcoplasmic reticulum; TG, transgenic; TnI, inhibitory subunit of troponin; WT, wild type; PKA, protein kinase A. are tightly and efficiently controlled in the cell and that abnormalities in PP function can cause or at least modify the course of diseases in human (1Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1589) Google Scholar). The main PP in the heart are serine/threonine phosphatases (reviewed in Ref. 2Herzig S. Neumann J. Physiol. Rev. 2000; 80: 173-210Crossref PubMed Scopus (244) Google Scholar). They are grouped mainly into PP 1, 2A, 2B, and 2C (3Nairn A.C. Shenolikar S. Curr. Opin. Neurobiol. 1992; 2: 296-301Crossref PubMed Scopus (56) Google Scholar). The main phosphatase activity in the heart is because of the catalytic subunits of PP1 and PP2A. More than a dozen PP1c (catalytic subunit of PP1) binding and regulatory proteins have been identified up to the present (2Herzig S. Neumann J. Physiol. Rev. 2000; 80: 173-210Crossref PubMed Scopus (244) Google Scholar). PP1c can bind to the cardiac RYR2 probably mediated via an additional protein, spinophilin (4Marks A.R. Marx S.O. Reiken S. Trends Cardiovasc. Med. 2002; 12: 166-170Crossref PubMed Scopus (80) Google Scholar). Holoenzymes of PP2A contain a 36-kDa catalytic subunit (PP2Ac) and a regulatory subunit of 65 kDa termed PR65 (A subunit). A third subunit, B, can be associated with the core structure (5Virshup D.M. Curr. Opin. Cell Biol. 2000; 12: 180-185Crossref PubMed Scopus (294) Google Scholar). Molecular cloning revealed the existence of two mammalian PP2Ac isoforms α and β, which share 97% identity in their primary sequence. Both isoforms are found in high levels in the heart. However, PP2Acα is about 10 times more abundant than the PP2Acβ form, probably because of a more active gene promoter (reviewed in Ref. 1Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1589) Google Scholar). The isoforms are encoded by different genes. PP2A is remarkably constant during evolution and may even be the most conserved of all known proteins (1Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1589) Google Scholar). Overexpression in mammalian cells has hitherto been unsuccessful. Only inactive PP2Ac has recently been expressed in a baculovirus system (6Myles T. Schmidt K. Evans D.R. Cron P. Hemmings B.A. Biochem. J. 2001; 357: 225-232Crossref PubMed Scopus (36) Google Scholar). A dominant negative mutant of PP2Aα, however, has been expressed in the brain of transgenic mice (7Kins S. Crameri A. Evans D.R. Hemmings B.A. Nitsch R.M. Gotz J. J. Biol. Chem. 2001; 276: 38193-38200Abstract Full Text Full Text PDF PubMed Google Scholar). The B subunits confer subcellular localization, developmental regulation, and cell specificity to the PP2A holoenzyme (at least in brain). The A subunit is a structural subunit that is tightly associated with PP2Ac forming a scaffold to which the appropriate B subunit can bind (1Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1589) Google Scholar, 2Herzig S. Neumann J. Physiol. Rev. 2000; 80: 173-210Crossref PubMed Scopus (244) Google Scholar). β-Adrenoreceptor stimulation leads to cAMP generation and to subsequent stimulation of cAMP-dependent protein kinases. The β-adrenoreceptor agonists increase the Ca2+ release from the SR by stimulating the Ca2+ influx through the L-type calcium channel. Relaxation of the muscle is because of the Ca2+ pump in the SR, called SERCA. β-Adrenergic catecholamines enhance the activity of SERCA in an indirect way. Enhanced phosphorylation of phospholamban (PLB), an intrinsic membrane protein of the cardiac SR abolishes the inhibition of SERCA and more Ca2+ is removed from the cytosol into the SR. During the next heartbeat more Ca2+ can be released from the filled SR store and more force is generated. Hence PLB phosphorylation greatly contributes to the positive inotropic and relaxant effect of β-adrenergic stimulation in the heart (8Simmerman H.K. Jones L.R. Physiol. Rev. 1998; 78: 921-947Crossref PubMed Scopus (470) Google Scholar). Increased phosphatase activity might contribute to cardiac hypertrophy and heart failure based on several independent lines of evidence. Increased mRNA expression and activity of PP1 has been noted in samples from patients suffering from end stage heart failure (9Neumann J. Eschenhagen T. Jones L.R. Linck B. Schmitz W. Scholz H. Zimmermann N. J. Mol. Cell. Cardiol. 1997; 29: 265-272Abstract Full Text PDF PubMed Scopus (217) Google Scholar, 10Mishra S. Gupta R.C. Tiwari N. Sharov V.G. Sabbah H.N. J. Heart Lung Transplant. 2002; 21: 366-373Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Increased phosphatase activity, if it is relevant, should lead to dephosphorylation of regulatory proteins, which has been reported by several independent laboratories: PLB (11Bartel S. Stein B. Eschenhagen T. Mende U. Neumann J. Schmitz W. Krause E.G. Karczewski P. Scholz H. Mol. Cell. Biochem. 1996; 157: 171-179Crossref PubMed Scopus (64) Google Scholar, 12Schwinger R.H. Munch G. Bolck B. Karczewski P. Krause E.G. Erdmann E. J. Mol. Cell. Cardiol. 1999; 31: 479-491Abstract Full Text PDF PubMed Scopus (248) Google Scholar) and troponin (Tn) I (13Bodor G.S. Oakeley A.E. Allen P.D. Crimmins D.L. Ladenson J.H. Anderson P.A. Circulation. 1997; 96: 1495-1500Crossref PubMed Scopus (195) Google Scholar, 14Zakhary D.R. Moravec C.S. Stewart R.W. Bond M. Circulation. 1999; 99: 505-510Crossref PubMed Scopus (120) Google Scholar) are dephosphorylated in ventricles from patients with end stage heart failure. A causal link between heart failure and PP1 is further strengthened by an animal model. Chronic β-adrenergic stimulation by infusion of isoproterenol leads to cardiac hypertrophy, increased PP activity, and PLB dephosphorylation (15Boknik P. Fockenbrock M. Herzig S. Knapp J. Linck B. Lüss H. Müller F.U. Müller T. Schmitz W. Schröder F. Neumann J. Naunyn-Schmiedebergs Arch. Pharmacol. 2000; 262: 222-231Google Scholar). This is a clinically relevant model of heart dysfunction because increased β-adrenergic stimulation is a hallmark of human heart failure and many heart failure patients can be successfully treated with β-adrenoreceptor blockers. Cardiac specific overexpression of the catalytic subunit of PP1 in the mouse led to depressed cardiac function, dilated cardiomyopathy, and premature mortality consistent with heart failure (16Carr A.N. Schmidt A.G. Suzuki Y. del Monte F. Sato Y. Lanner C. Breeden K. Jing S.L. Allen P.B. Greengard P. Yatani A. Hoit B.D. Grupp I.L. Hajjar R.J. DePaoli-Roach A.A. Kranias E.G. Mol. Cell. Biol. 2002; 22: 4124-4135Crossref PubMed Scopus (221) Google Scholar). There is another line of evidence for a deleterious function of PP2A in the heart. Fostriecin, which is a somewhat selective PP2A inhibitor, can reduce the infarct size in rabbit hearts (17Hastie C.J. Cohen P.T. FEBS Lett. 1998; 431: 357-361Crossref PubMed Scopus (99) Google Scholar, 18Weinbrenner C. Baines C.P. Liu G.S. Armstrong S.C. Ganote C.E. Walsh A.H. Honkanen R.E. Cohen M.V. Downey J.M. Circulation. 1998; 98: 899-905Crossref PubMed Scopus (75) Google Scholar). Based on these findings we hypothesized that increased expression of PP2A might, similarly as PP1c, alter cardiac function. We report here the generation of mice with cardiac specific overexpression of PP2A and followed the biochemical and physiological sequels to dissect the exact function of PPs in the mammalian heart. Isolation of cDNA Encoding Mouse PP2Acα—Total RNA was extracted from mouse hearts to the A of the the PP2Acα and of the was generated chain with and analysis was by an of enzyme mouse PP2Acα with and was from the and into the of a mouse cardiac α-myosin heavy chain promoter expression the The of the α-myosin heavy chain the protein for mouse PP2Acα and of and and the was from the as a and used for of mouse mice identified by and of was and used for further RNA from mice hearts was as of RNA from RNA the was used to the analysis 10 of RNA or of mRNA was by to and was with of the cDNA of mouse of samples in a in The at of 2; and 1; to the The was times for with a The was by for 10 at was the protein and and was The samples for 10 at of of protein to and in for at at treated with and for at or at followed by with different proteins and with in a and the The primary catalytic subunit, regulatory subunit from mouse regulatory subunit rabbit by rabbit rabbit rabbit by of mouse rabbit at or at mouse or by G. S. rabbit and rabbit from Cell phosphatase activity was with a as J. P. Herzig S. Schmitz W. Scholz H. Gupta R.C. J. Physiol. PubMed Google Scholar) with a as to of in of okadaic in to 10 of to a of 10 The for 10 at of a and to the for at the was on by of and of protein was by and the in the was in a the of transgenic and of with and and and of and and are in of hearts of mice on and in 10 and in a at an of about for 2 the in and to was used for all and with a and with binding with at with primary the catalytic subunit of PP2A of primary was with for with and with The of with primary and in the primary that been at for 2 with a of the and on a with appropriate for and for of the and into an and The and and as into for analysis on followed with on a shown are of independent that and on mice that with a of and a system with a for and and a for The and for The of the heart was from the as was the and the In of and The analysis of was by two to the mouse was in mice with of a the a size was into the a and at a of with the system The was for of the β-adrenoreceptor via the an pump Heart and the of and and of of from PP2A transgenic and wild of Ca2+ and of cell as U. Neumann J. F. U. Schmitz W. Jones L.R. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). The cells with the and at was at and the was at and The of at the two was used as an of Ca2+ of was with an The of was to cell and expressed as of Cell L-type the cell as U. Neumann J. F. U. Schmitz W. Jones L.R. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). that and Ca2+ by from a of to a of for 10 Cell and with an to are as between with at the Overexpression of PP2A in the Heart of mouse cDNA PP2Acα was from mouse hearts by and the was by This cDNA was into the expression of an α-myosin heavy chain promoter for generation of transgenic mice with of the transgene was PP2Ac expression in hearts from and wild mice was by and In from both RNA and mRNA we noted a of the transgene 1, A and cardiac was to to protein we noted an in PP2Acα This was as a increase and The activity and of PP2Ac can be by different regulatory we the expression of subunits and no between and mice in and protein expression and phosphorylation in mice from of mice at 10 or of to The to the of the specific protein in wild The abbreviations are used in the catalytic A regulatory regulatory PLB at PLB at are as of transgenic wild transgenic wild transgenic wild transgenic wild transgenic wild transgenic wild in a To if the PP2A was active or not we the phosphatase activity in heart with a as We found an phosphatase activity by To PP2A activity from PP1 activity we used 10 okadaic a known to PP2A not PP1 P. PubMed Scopus Google Scholar). the that the activity was mainly based on PP1 and PP2A activity, PP2A activity was as activity activity in the of 10 Whereas PP1 activity was between and PP2A activity was in the of PP2A to the protein phosphatase activity was from in hearts to in hearts To PP2Acα expression in the we of PP2Acα in and increased expression of PP2Acα in cardiac of We used and as well as and and isles of necrosis and of in the in and of in of contrast to PP2A, the expression of the catalytic subunit of PP1 remained with from mice that the protein expression of protein kinase A eukaryotic elongation factor 2 sarcoplasmic reticulum PLB, and the inhibitory subunit of remained in hearts and the phosphorylation state of the Ca2+ regulatory proteins PLB and as well as the phosphorylation state of was reduced in with revealed a phosphorylation of by A is in The phosphorylation state of PLB at and as by was reduced by and in ventricles and the phosphorylation state of was reduced by and and of and heart increased in mice by and This was accompanied by cellular hypertrophy cardiac hypertrophy may heart function we the of the transgene Cardiac revealed in the of mice In mice the of was by and the of was by with To the to β-adrenergic mice by infusion of a β-adrenergic Whereas the positive effect was the in developed and to β-adrenergic stimulation in mice the of the and by and in with and in via we and we by and by indicative of dilated and of function reduced as by a and a of for To the course of the function we cardiac on mice at and of Cardiac dilatation was present in Whereas of contractility between and and of reduced by and we noted a further of contractility at The dilatation of the was more between and up to of no noted for mice during the hypertrophy in mice heart and from wild and mice at 10 of The of was on of through the ventricles from and cells from of are as of in a of mice and wild of mice by of of are as of transgenic wild transgenic wild transgenic wild transgenic wild in a in wild and mice at of and of the in and mice the cardiac of of mice and wild with TG, PP2Acα transgenic WT, wild of as of as are as of 10 in a and L-type in Cardiac from Ca2+ and L-type Ca2+ of and from and β-adrenergic stimulation with The cell was reduced by in the of isoproterenol in with However, the to of was and in with The Ca2+ an increased Ca2+ by and the course of the of the Ca2+ was not by β-adrenergic stimulation in cells in contrast to cells cell revealed a of the L-type Ca2+ and in with the constant of as well as the constant of of the L-type Ca2+ and in with by by by by in and of from mice and wild and β-adrenergic stimulation with isoproterenol at a of The abbreviations are used in the to of the Ca2+ to of constant of constant of are as of cells from or Ca2+ in a that PP2A from animal and human heart J. 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Physiol. 2002; PubMed Scopus Google Scholar). a the that PP1 and the L-type Ca2+ PP2A the of J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, V. A. R.J. T. 2001; PubMed Scopus Google Scholar, J. Physiol. PubMed Scopus Google Scholar). overexpression of PP2Acα in a of and a of the L-type Ca2+ The increased PP2A activity in hearts was most the of PP2Acα as the expression of the and as well as which can alter PP2A activity, found PP2Acα may be increased in This should lead in to an PP the the phosphorylation state of is more than the phosphorylation state of or the expression is in this for of PP2Acα not all regulatory subunits in mice are to a catalytic subunit and can function as a of PP2A phosphatase activity a as was increased to a with the PP2Acα protein This may be by the binding of the protein to was shown that can bind to the catalytic subunit of PP2A with of binding of the of PP2A, with an increase in activity protein by protein to a K. J. D.L. U. S. 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In a model for the role of PP2A in the heart has been generated and as to cardiac hypertrophy and cardiac dilatation to human of heart failure. We Jones for SERCA and for Cell of for and G. S. for and We for