Superinhibition of Sarcoplasmic Reticulum Function by Phospholamban Induces Cardiac Contractile Failure
2001; Elsevier BV; Volume: 276; Issue: 26 Linguagem: Inglês
10.1074/jbc.m102403200
ISSN1083-351X
AutoresKobra Haghighi, Albrecht Schmidt, Brian D. Hoit, Angela G. Brittsan, Atsuko Yatani, James W. Lester, Jing Zhai, Yoshihiro Kimura, Gerald W. Dorn, David H. MacLennan, Evangelia G. Kranias,
Tópico(s)Cardiomyopathy and Myosin Studies
ResumoTo determine whether selective impairment of cardiac sarcoplasmic reticulum (SR) Ca2+ transport may drive the progressive functional deterioration leading to heart failure, transgenic mice, overexpressing a phospholamban Val49 → Gly mutant (2-fold), which is a superinhibitor of SR Ca2+-ATPase affinity for Ca2+, were generated, and their cardiac phenotype was examined longitudinally. At 3 months of age, the increased EC50 level of SR Ca2+ uptake for Ca2+ (0.67 ± 0.09 μm) resulted in significantly higher depression of cardiomyocyte rates of shortening (57%), relengthening (31%), and prolongation of the Ca2+ signal decay time (165%) than overexpression (2-fold) of wild type phospholamban (68%, 64%, and 125%, respectively), compared with controls (100%). Echocardiography also revealed significantly depressed function and impaired β-adrenergic responses in mutant hearts. The depressed contractile parameters were associated with left ventricular remodeling, recapitulation of fetal gene expression, and hypertrophy, which progressed to dilated cardiomyopathy with interstitial tissue fibrosis and death by 6 months in males. Females also had ventricular hypertrophy at 3 months but exhibited normal systolic function up to 12 months of age. These results suggest a causal relationship between defective SR Ca2+ cycling and cardiac remodeling leading to heart failure, with a gender-dependent influence on the time course of these alterations. To determine whether selective impairment of cardiac sarcoplasmic reticulum (SR) Ca2+ transport may drive the progressive functional deterioration leading to heart failure, transgenic mice, overexpressing a phospholamban Val49 → Gly mutant (2-fold), which is a superinhibitor of SR Ca2+-ATPase affinity for Ca2+, were generated, and their cardiac phenotype was examined longitudinally. At 3 months of age, the increased EC50 level of SR Ca2+ uptake for Ca2+ (0.67 ± 0.09 μm) resulted in significantly higher depression of cardiomyocyte rates of shortening (57%), relengthening (31%), and prolongation of the Ca2+ signal decay time (165%) than overexpression (2-fold) of wild type phospholamban (68%, 64%, and 125%, respectively), compared with controls (100%). Echocardiography also revealed significantly depressed function and impaired β-adrenergic responses in mutant hearts. The depressed contractile parameters were associated with left ventricular remodeling, recapitulation of fetal gene expression, and hypertrophy, which progressed to dilated cardiomyopathy with interstitial tissue fibrosis and death by 6 months in males. Females also had ventricular hypertrophy at 3 months but exhibited normal systolic function up to 12 months of age. These results suggest a causal relationship between defective SR Ca2+ cycling and cardiac remodeling leading to heart failure, with a gender-dependent influence on the time course of these alterations. sarcoplasmic reticulum phospholamban mutant phospholamban a mutant form of phospholamban in which Val49 is mutated to Gly cardiac sarcoplasmic reticulum, Ca2+-ATPase human embryonic kidney α- and β-myosin heavy chain atrial natriuretic peptide kilobase pair(s) L-type Ca2+ current left ventricular picofarads Cardiac hypertrophy and failure are highly complex disorders that arise as a result of a combination of mechanical, hemodynamic, hormonal, and pathological stimuli (1Levy D. Garrison R.J. Savage D.D. Kannel W.B. Castelli W.P. N. Engl. J. Med. 1990; 322: 1561-1566Crossref PubMed Scopus (4810) Google Scholar). In response to these effectors, the heart undergoes an adaptive response of compensatory hypertrophy (2Chien K.R. Cell. 1999; 98: 555-558Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar) followed by decompensated heart failure that is characterized by defects in Ca2+ handling during excitation-contraction coupling. Studies of end-stage-failing hearts have shown that the disturbed calcium homeostasis is associated with alterations in the expression levels or the activity of key Ca2+-handling proteins, leading to abnormal excitation contraction coupling and diastolic as well as systolic dysfunction (3Hasenfuss G. Cardiovasc. Res. 1998; 39: 60-76Crossref PubMed Scopus (338) Google Scholar, 4Houser S.R. Piacentino 3rd, V. Weisser J. J. Mol. Cell Cardiol. 2000; 32: 1595-1607Abstract Full Text PDF PubMed Scopus (278) Google Scholar). Specifically, alterations in SR1Ca2+-ATPase (SERCA2a) activity, the major Ca2+transport protein in SR, have been implicated as important determinants in the deteriorated function of the failing heart (5Meyer M. Schillinger W. Pieske B. Holubarsch C. Heilmann C. Posival H. Kuwajima G. Mikoshiba K. Just H. Hasenfuss G. Circulation. 1995; 92: 778-784Crossref PubMed Scopus (440) Google Scholar, 6Schmidt U. Hajjar R.J. Kim C.S. Lebeche D. Doye A.A. Gwathmey J.K. Am. J. Physiol. 1999; 277: H474-H480Crossref PubMed Google Scholar, 7Schwinger 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 (243) Google Scholar). The activity of SERCA2a is regulated by phospholamban (PLB), a 52-amino acid, muscle-specific SR phosphoprotein (8Koss K.L. Kranias E.G. Circ. Res. 1996; 79: 1059-1063Crossref PubMed Scopus (288) Google Scholar, 9Simmerman H.K. Jones L.R. Physiol. Rev. 1998; 78: 921-947Crossref PubMed Scopus (461) Google Scholar, 10Tada M. Yabuki M. Toyofuku T. Ann. N. Y. Acad. Sci. 1998; 853: 116-129Crossref PubMed Scopus (12) Google Scholar). Dephosphorylated PLB inhibits the Ca2+ affinity of SERCA2a, whereas the phosphorylated form of PLB dissociates from SERCA2a leading to increases in Ca2+ uptake rates and accelerated ventricular relaxation (11Luo W. Chu G. Sato Y. Zhou Z. Kadambi V.J. Kranias E.G. J. Biol. Chem. 1998; 273: 4734-4739Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 12Wegener A.D. Simmerman H.K. Lindemann J.P. Jones L.R. J. Biol. Chem. 1989; 264: 11468-11474Abstract Full Text PDF PubMed Google Scholar, 13Tada M. Yamada M. Kadoma M. Inui M. Ohmori F. Mol. Cell Biochem. 1982; 46: 73-95Crossref PubMed Scopus (72) Google Scholar).The role of PLB in the regulation of basal contractility has been elucidated through the development of genetically engineered mouse models. Phospholamban ablation resulted in significant increases in cardiac contractile parameters, whereas overexpressing PLB was associated with depressed cardiac function (14Kadambi V.J. Ponniah S. Harrer J.M. Hoit B.D. Dorn 2nd, G.W. Walsh R.A. Kranias E.G. J. Clin. Invest. 1996; 97: 533-539Crossref PubMed Scopus (275) Google Scholar, 15Luo W. Grupp I.L. Harrer J. Ponniah S. Grupp G. Duffy J.J. Doetschman T. Kranias E.G. Circ. Res. 1994; 75: 401-409Crossref PubMed Scopus (627) Google Scholar). Actually, an inverse relationship between the ratio of PLB/SERCA2a and myocardial contractility was observed (16Koss K.L. Grupp I.L. Kranias E.G. Basic Res. Cardiol. 1997; 92: 17-24Crossref PubMed Google Scholar), suggesting that any process that alters the relative levels of these proteins and/or the interaction between them may result in altered myocardial contractility. However, it is not currently known whether a primary defect in SR Ca2+ uptake, elicited by altered interactions between PLB and SERCA2a, may result in depressed cellular Ca2+homeostasis and drive the progressive deterioration of function leading to heart failure.Early studies evaluating the interaction between PLB and SERCA2a were directed toward the cytoplasmic domain (amino acid residues 1–30), which includes the phosphorylation sites in PLB (17Toyofuku T. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 1994; 269: 3088-3094Abstract Full Text PDF PubMed Google Scholar, 18Jones L.R. Field L.J. J. Biol. Chem. 1993; 268: 11486-11488Abstract Full Text PDF PubMed Google Scholar). More recently, the transmembrane interaction sites (amino acid residues 31–52) were shown to mediate the regulatory effects of PLB on SERCA2a affinity, with some mutations yielding increased inhibition, whereas others abolish the PLB inhibitory effects on SERCA2a in vitro (19Kimura Y. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 1996; 271: 21726-21731Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 20Kimura Y. Asahi M. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 1998; 273: 14238-14241Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 21Cornea R.L. Autry J.M. Chen Z. Jones L.R. J. Biol. Chem. 2000; 275: 41487-41494Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Extension of these studies to in vivomodels indicated that overexpression of either L37A or I40A mutant PLB resulted in depressed cardiac myocyte Ca2+ kinetics and mechanical parameters associated with hypertrophy (22Zvaritch E. Backx P.H. Jirik F. Kimura Y. de Leon S. Schmidt A.G. Hoit B.D. Lester J.W. Kranias E.G. MacLennan D.H. J. Biol. Chem. 2000; 275: 14985-14991Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Further support for the functional significance of the PLB transmembrane domain in mediating its regulatory effects on SERCA2a was provided by a recent study, in which a Val49 → Ala (V49A) mutant PLB was expressed in vivo, using recombinant adenoviruses. The V49A mutant acted dominantly to increase contractility in cardiac ventricular cells in the absence of catecholamines (23Minamisawa S. Hoshijima M. Chu G. Ward C.A. Frank K. Gu Y. Martone M.E. Wang Y. Ross Jr., J. Kranias E.G. Giles W.R. Chien K.R. Cell. 1999; 99: 313-322Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar). This study pointed out the importance of the PLB carboxyl terminus in mediating its interaction with SERCA2a and proposed that interfering with this interaction may provide a novel therapeutic approach for the prevention of dilated cardiomyopathy. To further elucidate the functional significance of the PLB Val49 residue in vivo, we generated a mutant, Val49 → Gly (V49G), which acts as a potent inhibitor of the Ca2+ affinity of SERCA2a. Cardiac-specific overexpression of the V49G mutant PLB resulted in superinhibition of cardiac contractility and cardiac remodeling, which progressed to dilated cardiomyopathy and early mortality. Thus, enhanced inhibition of SERCA2a by PLB may serve as a prime candidate for driving the onset and progression of heart failure.DISCUSSIONThis is the first study to demonstrate that a mutation in PLB, which is associated with superinhibition of SERCA2a and contractile parameters, may lead to dilated cardiomyopathy, overt heart failure, and early mortality, which are modified by gender. Previous studies in animal models or human with end stage heart failure showed that the increases in diastolic calcium and impaired relaxation were linked to inhibition of SR Ca2+ transport (3Hasenfuss G. Cardiovasc. Res. 1998; 39: 60-76Crossref PubMed Scopus (338) Google Scholar, 29Morgan J.P. N. Engl. J. Med. 1991; 325: 625-632Crossref PubMed Scopus (274) Google Scholar). However, the depressed SR function was suggested to be secondary to insults arising from extracellular effectors and may act as a modifier for the progression of cardiac deterioration (30Chien K.R. J. Clin. Invest. 2000; 105: 1339-1342Crossref PubMed Scopus (55) Google Scholar). Thus, it is not currently clear whether inhibition of SR Ca2+ transport is sufficient to cause heart failure or it is a contributing factor to cardiomyocyte dysfunction in the context of pre-existing heart disease. To better address the role of depressed SR Ca2+ transport activity in the onset and progression of heart failure, we generated a transgenic model harboring the V49G mutation in PLB, which we identified as a superinhibitor of SERCA2a Ca2+ affinity in vitro, and studied the cardiac phenotype over the life span of the mouse. Overexpression of this mutant PLB was associated with significant depression in SR Ca2+ uptake rates. The inhibited SR function resulted in impaired cardiac contractile parameters in isolated cardiomyocytes and intact mice at 3 months of age. In cardiomyocytes, the attenuated mechanical parameters reflected a significant prolongation in the rate of the Ca2+ signal decay (T80) without alteration in the peak amplitude of the Ca2+ transient. The lack of peak amplitude alteration may reflect increased Ca2+ influx through the L-type Ca2+ channels, whose density was increased in transgenic myocytes, indicating an important compensatory response in an attempt to normalize systolic Ca2+ levels in these cells. The V49G mutant myocytes also exhibited significantly slower ICa inactivation, despite increased Ca2+ influx through the L-type Ca2+ channels, suggesting that the SR Ca2+ release was diminished (26Masaki H. Sato Y. Luo W. Kranias E.G. Yatani A. Am. J. Physiol. 1997; 272: H606-H612PubMed Google Scholar, 31Sham J.S. Cleemann L. Morad M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 121-125Crossref PubMed Scopus (206) Google Scholar). Importantly, similar phenotypic alterations were exhibited in a second line (line 4), which expressed the same levels of mutant PLB, indicating that the inhibitory effect was due to transgene expression and not any possible insertional mutation.The mechanisms underlying the superinhibitory effects of V49G mutant PLB in vivo are not clear. However, this study together with a previous one, utilizing a V49A mutant (23Minamisawa S. Hoshijima M. Chu G. Ward C.A. Frank K. Gu Y. Martone M.E. Wang Y. Ross Jr., J. Kranias E.G. Giles W.R. Chien K.R. Cell. 1999; 99: 313-322Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar), emphasize that the Val49 residue in PLB, which is highly conserved among species, is important for the PLB/SERCA2a interaction and changes in this region have profound effects on SR function and cardiac contractility. Thus, the V49G mutation, employed in this study, may influence the three-dimensional structure of PLB and enhance its interaction with SERCA2a. Importantly, the normal ratio (10:1) of pentamers to monomers was not altered by this mutation in PLB, further supporting that the superinhibitory effect of the mutant may be due to its conformational change. An enhanced association between PLB and SERCA2a would be consistent with the inability of isoproterenol to fully relieve the V49G mutant PLB superinhibitory effects, similar to previous findings with the N27A PLB mutant (24Zhai J. Schmidt A.G. Hoit B.D. Kimura Y. MacLennan D.H. Kranias E.G. J. Biol. Chem. 2000; 275: 10538-10544Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). In contrast, the reduced contractility in L37A and I40A PLB mutant mice could be completely over come by isoproterenol stimulation (22Zvaritch E. Backx P.H. Jirik F. Kimura Y. de Leon S. Schmidt A.G. Hoit B.D. Lester J.W. Kranias E.G. MacLennan D.H. J. Biol. Chem. 2000; 275: 14985-14991Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar).It is of interest that cardiac-specific overexpression of the PLB V49G mutant induced cardiac hypertrophy in an attempt to normalize cardiac function (32Lorell B.H. Carabello B.A. Circulation. 2000; 102: 470-479Crossref PubMed Scopus (828) Google Scholar, 33Grossman W. Am. J. Med. 1980; 69: 576-584Abstract Full Text PDF PubMed Scopus (413) Google Scholar), which was severely depressed in mutant hearts. However, the molecular mechanisms that link depressed SR Ca2+ cycling to reprogramming of gene expression in the PLB V49G hearts are not clear. Hypertrophy was associated with induction or re-expression of a fetal gene program, including ANF, β-myosin heavy chain, and α-skeletal actin in these hearts (34Feldman A.M. Weinberg E.O. Ray P.E. Lorell B.H. Circ. Res. 1993; 73: 184-192Crossref PubMed Scopus (319) Google Scholar). The increases in the protein expression levels of the slow myosin heavy chain (β-MHC) isoform (35Bristow M.R. Lancet. 1998; 352 Suppl. 1: SI8-SI14Abstract Full Text Full Text PDF PubMed Google Scholar) may also contribute to the observed attenuation of contraction and relaxation rates in the mutant PLB hearts. Interestingly, the hypertrophic remodeling, which provided an initial compensatory phase, progressed to cardiac failure and premature death in this model expressing the V49G PLB superinhibitor. Previous studies on the N27A (24Zhai J. Schmidt A.G. Hoit B.D. Kimura Y. MacLennan D.H. Kranias E.G. J. Biol. Chem. 2000; 275: 10538-10544Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) and L37A and I40A (22Zvaritch E. Backx P.H. Jirik F. Kimura Y. de Leon S. Schmidt A.G. Hoit B.D. Lester J.W. Kranias E.G. MacLennan D.H. J. Biol. Chem. 2000; 275: 14985-14991Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) PLB superinhibitors also showed severely depressed SR and cardiac function in vivo, which were associated with hypertrophy by 3 months of age. However, the hypertrophic phenotype of the N27A, L37A, and I40A mutant mice did not progress to overt heart failure, as observed with the V49G mutant. The apparent differences between these transgenic models may be due to the expression levels of the various PLB mutants in vivo, the age of the mice studied, and/or the time course of transition between the compensatory hypertrophic response and heart failure. In addition, there may be differences in activated intrinsic hypertrophic signaling pathways, triggered by SR dysfunction, which lead to different courses of myocyte maladaptation among these transgenic mice. However, the present findings and those in other models (22Zvaritch E. Backx P.H. Jirik F. Kimura Y. de Leon S. Schmidt A.G. Hoit B.D. Lester J.W. Kranias E.G. MacLennan D.H. J. Biol. Chem. 2000; 275: 14985-14991Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 23Minamisawa S. Hoshijima M. Chu G. Ward C.A. Frank K. Gu Y. Martone M.E. Wang Y. Ross Jr., J. Kranias E.G. Giles W.R. Chien K.R. Cell. 1999; 99: 313-322Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 24Zhai J. Schmidt A.G. Hoit B.D. Kimura Y. MacLennan D.H. Kranias E.G. J. Biol. Chem. 2000; 275: 10538-10544Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 37Kadokami T. McTiernan C.F. Kubota T. Frye C.S. Feldman A.M. J. Clin. Invest. 2000; 106: 589-597Crossref PubMed Scopus (112) Google Scholar) suggest that multiple pathways, including disturbed SR Ca2+ homeostasis, may operate in concert to induce a hypertrophic response leading to heart failure.Several studies have indicated that cardiovascular mortality is higher in males than in females, which frequently exhibit preserved cardiac performance, and suggested a gender-dependent influence on the onset and progression of heart failure (36Adams Jr., K.F. Sueta C.A. Gheorghiade M. O'Connor C.M. Schwartz T.A. Koch G.G. Uretsky B. Swedberg K. McKenna W. Soler-Soler J. Califf R.M. Circulation. 1999; 99: 1816-1821Crossref PubMed Google Scholar). In the present study, we also observed an early mortality in males (6 months) compared with females (15 months) in our model with genetically induced SR Ca2+-handling defects. The striking survival benefit in females was not due to differences in their levels of PLB, compared with age-matched males. Therefore, we investigated gender-specific differences in cardiac function and in the progression to heart failure. Males and females were examined during compensatory hypertrophy and after the appearance of symptoms of heart failure, using echocardiography. LV-to-body mass was about 50% increased by 3 months of age, and cardiac function was significantly depressed in male PLB mutant mice. The remodeling process accelerated the progression to heart failure, characterized by significant chamber dilation and a decrease in the ratio of wall thickness to chamber diameter (h/r) by 6 months of age. However, the progression to failure was delayed in females (37Kadokami T. McTiernan C.F. Kubota T. Frye C.S. Feldman A.M. J. Clin. Invest. 2000; 106: 589-597Crossref PubMed Scopus (112) Google Scholar), indicating that, despite a similar degree of LV hypertrophy between males and females, there were significant gender differences in the LV-adaptive response to pathological hypertrophy and depressed function, similar to recent studies in the TNF-α overexpression model and in a rat model of pressure overload hypertrophy (38Weinberg E.O. Thienelt C.D. Katz S.E. Bartunek J. Tajima M. Rohrbach S. Douglas P.S. Lorell B.H. J. Am. Coll. Cardiol. 1999; 34: 264-273Crossref PubMed Scopus (201) Google Scholar). The etiology of these differences is unclear but may be related to: (a) a reduced adaptive hypertrophic reserve in males (39Weinberg E.O. Lee M.A. Weigner M. Lindpaintner K. Bishop S.P. Benedict C.R. Ho K.K. Douglas P.S. Chafizadeh E. Lorell B.H. Circulation. 1997; 95: 1592-1600Crossref PubMed Scopus (79) Google Scholar); (b) the lower mitochondria respiratory and lysosomal enzyme activity in females (40Koenig H. Goldstone A. Lu C.Y. Circ. Res. 1982; 50: 782-787Crossref PubMed Scopus (94) Google Scholar); (c) a higher percentage of the V1 myosin isozyme, which is up-regulated by estrogen in females (41Schaible T.F. Malhotra A. Ciambrone G. Scheuer J. Circ. Res. 1984; 54: 38-49Crossref PubMed Scopus (156) Google Scholar); and (d) the intrinsic gender-specific differences in cardiac muscle physiology and biochemistry (42Buttrick P. Scheuer J. Circulation. 1992; 86: 1336-1338Crossref PubMed Scopus (26) Google Scholar). Furthermore, estrogen signaling through the adult myocyte estrogen receptor may contribute to gender differences in gene expression in pathological hypertrophy (43Grohe C. Kahlert S. Lobbert K. Stimpel M. Karas R.H. Vetter H. Neyses L. FEBS Lett. 1997; 416: 107-112Crossref PubMed Scopus (319) Google Scholar). Interestingly, in humans with congenital aortic stenosis and cardiac hypertrophy, there appears to be an overcompensation of myocardial contraction early on (44Donner R. Carabello B.A. Black I. Spann J.F. Am. J. Cardiol. 1983; 51: 946-951Abstract Full Text PDF PubMed Scopus (39) Google Scholar), which is similar to that described in mutant PLB females in this study.In summary, our findings point to a primary defect in SR function as an inducer of a phenotype that resembles human dilated cardiomyopathy and ventricular remodeling. The enhanced inhibition of SERCA2a by the V49G mutant PLB, associated with depressed myocyte calcium homeostasis, resulted in a remodeling process, which involved interaction between all components of the myocardium leading to overt heart failure. Furthermore, gender significantly influenced the evolution of the early responses to LV remodeling, including the transition to heart failure in the mutant PLB model. Future studies on the cellular and molecular mechanisms underlying these responses, using gene expression profiling and proteomics, may unveil specific molecules or pathways by which depressed SR function influences the onset of hypertrophy and the transition to dilated cardiomyopathy in this model. Cardiac hypertrophy and failure are highly complex disorders that arise as a result of a combination of mechanical, hemodynamic, hormonal, and pathological stimuli (1Levy D. Garrison R.J. Savage D.D. Kannel W.B. Castelli W.P. N. Engl. J. Med. 1990; 322: 1561-1566Crossref PubMed Scopus (4810) Google Scholar). In response to these effectors, the heart undergoes an adaptive response of compensatory hypertrophy (2Chien K.R. Cell. 1999; 98: 555-558Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar) followed by decompensated heart failure that is characterized by defects in Ca2+ handling during excitation-contraction coupling. Studies of end-stage-failing hearts have shown that the disturbed calcium homeostasis is associated with alterations in the expression levels or the activity of key Ca2+-handling proteins, leading to abnormal excitation contraction coupling and diastolic as well as systolic dysfunction (3Hasenfuss G. Cardiovasc. Res. 1998; 39: 60-76Crossref PubMed Scopus (338) Google Scholar, 4Houser S.R. Piacentino 3rd, V. Weisser J. J. Mol. Cell Cardiol. 2000; 32: 1595-1607Abstract Full Text PDF PubMed Scopus (278) Google Scholar). Specifically, alterations in SR1Ca2+-ATPase (SERCA2a) activity, the major Ca2+transport protein in SR, have been implicated as important determinants in the deteriorated function of the failing heart (5Meyer M. Schillinger W. Pieske B. Holubarsch C. Heilmann C. Posival H. Kuwajima G. Mikoshiba K. Just H. Hasenfuss G. Circulation. 1995; 92: 778-784Crossref PubMed Scopus (440) Google Scholar, 6Schmidt U. Hajjar R.J. Kim C.S. Lebeche D. Doye A.A. Gwathmey J.K. Am. J. Physiol. 1999; 277: H474-H480Crossref PubMed Google Scholar, 7Schwinger 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 (243) Google Scholar). The activity of SERCA2a is regulated by phospholamban (PLB), a 52-amino acid, muscle-specific SR phosphoprotein (8Koss K.L. Kranias E.G. Circ. Res. 1996; 79: 1059-1063Crossref PubMed Scopus (288) Google Scholar, 9Simmerman H.K. Jones L.R. Physiol. Rev. 1998; 78: 921-947Crossref PubMed Scopus (461) Google Scholar, 10Tada M. Yabuki M. Toyofuku T. Ann. N. Y. Acad. Sci. 1998; 853: 116-129Crossref PubMed Scopus (12) Google Scholar). Dephosphorylated PLB inhibits the Ca2+ affinity of SERCA2a, whereas the phosphorylated form of PLB dissociates from SERCA2a leading to increases in Ca2+ uptake rates and accelerated ventricular relaxation (11Luo W. Chu G. Sato Y. Zhou Z. Kadambi V.J. Kranias E.G. J. Biol. Chem. 1998; 273: 4734-4739Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 12Wegener A.D. Simmerman H.K. Lindemann J.P. Jones L.R. J. Biol. Chem. 1989; 264: 11468-11474Abstract Full Text PDF PubMed Google Scholar, 13Tada M. Yamada M. Kadoma M. Inui M. Ohmori F. Mol. Cell Biochem. 1982; 46: 73-95Crossref PubMed Scopus (72) Google Scholar). The role of PLB in the regulation of basal contractility has been elucidated through the development of genetically engineered mouse models. Phospholamban ablation resulted in significant increases in cardiac contractile parameters, whereas overexpressing PLB was associated with depressed cardiac function (14Kadambi V.J. Ponniah S. Harrer J.M. Hoit B.D. Dorn 2nd, G.W. Walsh R.A. Kranias E.G. J. Clin. Invest. 1996; 97: 533-539Crossref PubMed Scopus (275) Google Scholar, 15Luo W. Grupp I.L. Harrer J. Ponniah S. Grupp G. Duffy J.J. Doetschman T. Kranias E.G. Circ. Res. 1994; 75: 401-409Crossref PubMed Scopus (627) Google Scholar). Actually, an inverse relationship between the ratio of PLB/SERCA2a and myocardial contractility was observed (16Koss K.L. Grupp I.L. Kranias E.G. Basic Res. Cardiol. 1997; 92: 17-24Crossref PubMed Google Scholar), suggesting that any process that alters the relative levels of these proteins and/or the interaction between them may result in altered myocardial contractility. However, it is not currently known whether a primary defect in SR Ca2+ uptake, elicited by altered interactions between PLB and SERCA2a, may result in depressed cellular Ca2+homeostasis and drive the progressive deterioration of function leading to heart failure. Early studies evaluating the interaction between PLB and SERCA2a were directed toward the cytoplasmic domain (amino acid residues 1–30), which includes the phosphorylation sites in PLB (17Toyofuku T. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 1994; 269: 3088-3094Abstract Full Text PDF PubMed Google Scholar, 18Jones L.R. Field L.J. J. Biol. Chem. 1993; 268: 11486-11488Abstract Full Text PDF PubMed Google Scholar). More recently, the transmembrane interaction sites (amino acid residues 31–52) were shown to mediate the regulatory effects of PLB on SERCA2a affinity, with some mutations yielding increased inhibition, whereas others abolish the PLB inhibitory effects on SERCA2a in vitro (19Kimura Y. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 1996; 271: 21726-21731Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 20Kimura Y. Asahi M. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 1998; 273: 14238-14241Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 21Cornea R.L. Autry J.M. Chen Z. Jones L.R. J. Biol. Chem. 2000; 275: 41487-41494Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Extension of these studies to in vivomodels indicated that overexpression of either L37A or I40A mutant PLB resulted in depressed cardiac myocyte Ca2+ kinetics and mechanical parameters associated with hypertrophy (22Zvaritch E. Backx P.H. Jirik F. Kimura Y. de Leon S. Schmidt A.G. Hoit B.D. Lester J.W. Kranias E.G. MacLennan D.H. J. Biol. Chem. 2000; 275: 14985-14991Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Further support for the functional significance of the PLB transmembrane domain in mediating its regulatory effects on SERCA2a was provided by a recent study, in which a Val49 → Ala (V49A) mutant PLB was expressed in vivo, using recombinant adenoviruses. The V49A mutant acted dominantly to increase contractility in cardiac ventricular cells in the absence of catecholamines (23Minamisawa S. Hoshijima M. Chu G. Ward C.A. Frank K. Gu Y. Martone M.E. Wang Y. Ross Jr., J. Kranias E.G. Giles W.R. Chien K.R. Cell. 1999; 99: 313-322Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar). This study pointed out the importance of the PLB carboxyl terminus in mediating its interaction with SERCA2a and proposed that interfering with this interaction may provide a novel therapeutic approach for the prevention of dilated cardiomyopathy. To further elucidate the functional significance of the PLB Val49 residue in vivo, we generated a mutant, Val49 → Gly (V49G), which acts as a potent inhibitor of the Ca2+ affinity of SERCA2a. Cardiac-specific overexpression of the V49G mutant PLB resulted in superinhibition of cardiac con
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