Cardiac-specific Overexpression of Mouse Cardiac Calsequestrin Is Associated with Depressed Cardiovascular Function and Hypertrophy in Transgenic Mice
1998; Elsevier BV; Volume: 273; Issue: 43 Linguagem: Inglês
10.1074/jbc.273.43.28470
ISSN1083-351X
AutoresYoji Sato, Donald G. Ferguson, Hidenori Sako, Gerald W. Dorn, Vivek J. Kadambi, Atsuko Yatani, Brian D. Hoit, Richard A. Walsh, Evangelia G. Kranias,
Tópico(s)Ion channel regulation and function
ResumoCalsequestrin is a high capacity Ca2+-binding protein in the sarcoplasmic reticulum (SR) lumen. To elucidate the functional role of calsequestrin in vivo, transgenic mice were generated that overexpressed mouse cardiac calsequestrin in the heart. Overexpression (20-fold) of calsequestrin was associated with cardiac hypertrophy and induction of a fetal gene expression program. Isolated transgenic cardiomyocytes exhibited diminished shortening fraction (46%), shortening rate (60%), and relengthening rate (60%). The Ca2+ transient amplitude was also depressed (45%), although the SR Ca2+storage capacity was augmented, as suggested by caffeine application studies. These alterations were associated with a decrease in L-type Ca2+ current density and prolongation of this channel's inactivation kinetics without changes in Na+-Ca2+ exchanger current density. Furthermore, there were increases in protein levels of SR Ca2+-ATPase, phospholamban, and calreticulin and decreases in FKBP12, without alterations in ryanodine receptor, junctin, and triadin levels in transgenic hearts. Left ventricular function analysis in Langendorff perfused hearts and closed-chest anesthetized mice also indicated depressed rates of contraction and relaxation of transgenic hearts. These findings suggest that calsequestrin overexpression is associated with increases in SR Ca2+ capacity, but decreases in Ca2+-induced SR Ca2+ release, leading to depressed contractility in the mammalian heart. Calsequestrin is a high capacity Ca2+-binding protein in the sarcoplasmic reticulum (SR) lumen. To elucidate the functional role of calsequestrin in vivo, transgenic mice were generated that overexpressed mouse cardiac calsequestrin in the heart. Overexpression (20-fold) of calsequestrin was associated with cardiac hypertrophy and induction of a fetal gene expression program. Isolated transgenic cardiomyocytes exhibited diminished shortening fraction (46%), shortening rate (60%), and relengthening rate (60%). The Ca2+ transient amplitude was also depressed (45%), although the SR Ca2+storage capacity was augmented, as suggested by caffeine application studies. These alterations were associated with a decrease in L-type Ca2+ current density and prolongation of this channel's inactivation kinetics without changes in Na+-Ca2+ exchanger current density. Furthermore, there were increases in protein levels of SR Ca2+-ATPase, phospholamban, and calreticulin and decreases in FKBP12, without alterations in ryanodine receptor, junctin, and triadin levels in transgenic hearts. Left ventricular function analysis in Langendorff perfused hearts and closed-chest anesthetized mice also indicated depressed rates of contraction and relaxation of transgenic hearts. These findings suggest that calsequestrin overexpression is associated with increases in SR Ca2+ capacity, but decreases in Ca2+-induced SR Ca2+ release, leading to depressed contractility in the mammalian heart. sarcoplasmic reticulum base pair(s) myosin heavy chain L-type Ca2+ current picofarad(s). In cardiac excitation-contraction coupling, the sarcoplasmic reticulum (SR)1 plays an essential role in the regulation of the cytosolic free Ca2+concentration. There are three major functions of the SR: (a) Ca2+ uptake from the cytosol into the SR lumen, resulting in muscle relaxation; (b) Ca2+storage in the SR lumen; and (c) Ca2+ release from the SR into the cytosol, resulting in muscle contraction. The main SR proteins responsible for these functions are the Ca2+transport ATPase, the Ca2+ storage protein calsequestrin (1Yano K. Zarain-Herzberg A. Mol. Cell. Biochem. 1994; 135: 61-70Crossref PubMed Scopus (137) Google Scholar), and the Ca2+ release channel or ryanodine receptor, respectively. Phospholamban is another SR protein that plays a crucial role in the modulation of myocardial contractility and relaxation (2Koss K.L. Kranias E.G. Circ. Res. 1996; 79: 1059-1063Crossref PubMed Scopus (288) Google Scholar).Recent studies with isolated human myocardial preparations suggested that the impaired Ca2+ handling of the SR may be an important subcellular mechanism contributing to the depressed contractility in heart failure (3Hasenfuss G. Reinecke H. Studer R. Pieske B. Meyer M. Drexler H. Just H. Basic Res. Cardiol. 1996; 91 Suppl. 2: 17-22Crossref PubMed Google Scholar, 4Wankerl M. Schwartz K. J. Mol. Med. 1995; 73: 487-496Crossref PubMed Scopus (55) Google Scholar, 5Beuckelmann D.J. Nabauer M. Erdmann E. Circulation. 1992; 85: 1046-1055Crossref PubMed Scopus (720) Google Scholar). However, it is still controversial whether the expression levels of SR Ca2+-handling proteins are altered in failing human hearts. Some groups have reported alterations in mRNA levels and/or protein expression levels of SR Ca2+-ATPase, phospholamban, and the SR Ca2+ release channel in human heart failure (3Hasenfuss G. Reinecke H. Studer R. Pieske B. Meyer M. Drexler H. Just H. Basic Res. Cardiol. 1996; 91 Suppl. 2: 17-22Crossref PubMed Google Scholar, 6Hasenfuss G. Meyer M. Schillinger W. Preuss M. Pieske B. Just H. Basic Res. Cardiol. 1997; 92 Suppl. 1: 87-93Crossref PubMed Google Scholar, 7Arai M. Alpert N.R. MacLennan D.H. Barton P. Periasamy M. Circ. Res. 1993; 72: 463-469Crossref PubMed Scopus (433) Google Scholar). However, other groups observed no significant changes in the expression levels of these proteins in hearts with end-stage heart failure (8Movsesian M.A. Karimi M. Green K. Jones L.R. Circulation. 1994; 90: 653-657Crossref PubMed Scopus (212) Google Scholar, 9Schwinger R.H.G. Böhm M. Schmidt U. Karczewski P. Bavendiek U. Flesch M. Krause E.G. Erdmann E. Circulation. 1995; 92: 3220-3228Crossref PubMed Scopus (346) Google Scholar). Interestingly, in all studies, the calsequestrin expression levels appeared to be unaltered. These findings suggest that calsequestrin expression is under specific and rigid regulation in cardiac muscle. However, the physiological significance of the apparent tight control of calsequestrin expression is not known.There are currently two different calsequestrin genes, encoding for the cardiac and fast-skeletal muscle products. The cardiac isoform of calsequestrin is highly conserved among species (1Yano K. Zarain-Herzberg A. Mol. Cell. Biochem. 1994; 135: 61-70Crossref PubMed Scopus (137) Google Scholar), and it is the only isoform expressed during cardiac development (10Mahony L. Jones L.R. J. Biol. Chem. 1986; 261: 15257-15265Abstract Full Text PDF PubMed Google Scholar, 11Arai M. Otsu K. MacLennan D.H. Periasamy M. Am. J. Physiol. 1992; 262: C614-C620Crossref PubMed Google Scholar). Several laboratories (for review, see Ref. 12Sitsapesan R. Williams A.J. J. Membr. Biol. 1997; 159: 179-185Crossref PubMed Scopus (115) Google Scholar) have implicated calsequestrin, which binds Ca2+ with high capacity and low affinity, as a key player in the regulation of SR Ca2+ release. Actually, calsequestrin appears to be physically connected to the ryanodine receptor (13Franzini-Armstrong C. Protasi F. Physiol. Rev. 1997; 77: 699-729Crossref PubMed Scopus (591) Google Scholar), and formation of a stable complex among calsequestrin, the ryanodine receptor, junctin, and triadin at the junctional SR has been proposed to be important for the operation of Ca2+release during cardiac muscle excitation-contraction coupling (14Zhang L. Kelley J. Schmeisser G. Kobayashi Y.M. Jones L.R. J. Biol. Chem. 1997; 272: 23389-23397Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar). However, it is not currently clear how calsequestrin buffers luminal Ca2+ in vivo, what is the buffering capacity of this protein in intact cells, and what are the direct or indirect effects of altered calsequestrin levels on SR Ca2+ load, ryanodine receptor gating, and myocardial contractility. Furthermore, the role of calsequestrin in the decreased amplitude of the intracellular Ca2+ transient in failing hearts, which may reflect a reduction in the amount of releasable Ca2+ from the SR (5Beuckelmann D.J. Nabauer M. Erdmann E. Circulation. 1992; 85: 1046-1055Crossref PubMed Scopus (720) Google Scholar, 15Vahl C.F. Bonz A. Timek T. Hagl S. Circ. Res. 1994; 74: 952-958Crossref PubMed Scopus (71) Google Scholar, 16Gwathmey J.K. Bentivegna L.A. Ransil B.J. Grossman W. Morgan J.P. Cardiovasc. Res. 1993; 27: 199-203Crossref PubMed Scopus (38) Google Scholar), is not clear. Thus, this study was designed to elucidate the functional role of calsequestrin in cardiac physiology and/or pathophysiology by altering the expression levels of this protein in vivo using transgenesis. Cardiac-specific overexpression of calsequestrin resulted in increased SR Ca2+ storage capacity, but this SR Ca2+ was not available for release during excitation-contraction coupling, leading to a depressed amplitude of the Ca2+ transient in cardiomyocytes and depressed contractile parameters assessed in isolated myocytes, perfused hearts, and intact animals. This cardiac phenotype was accompanied by alterations in the expression levels of several key proteins and induction of a fetal gene program leading to cardiac hypertrophy.EXPERIMENTAL PROCEDURESThe handling and maintenance of the animals in this study were approved by the ethics committee of the University of Cincinnati. 8–13-week-old mice of either sex were used for the following studies.Isolation of cDNA Encoding Mouse Cardiac CalsequestrinThe mouse cardiac 5′-stretch plus cDNA library (ML5002a; CLONTECH) was screened using a32P-labeled polymerase chain reaction product by a pUC/M13 forward primer (24-mer; Promega), a reverse primer corresponding to 60 nucleotides in the rabbit cardiac calsequestrin cDNA sequence (17Arai M. Alpert N.R. Periasamy M. Gene (Amst.). 1991; 109: 275-279Crossref PubMed Scopus (44) Google Scholar,18Chu G. Luo W. Slack J.P. Tilgman C. Sweet W.E. Spindle M. Saupe K.W. Boivin G.P. Moravec C.S. Matlib M.A. Grupp I.L. Ingwall J.S. Kranias E.G. Circ. Res. 1996; 79: 1064-1076Crossref PubMed Scopus (110) Google Scholar), and the rabbit cardiac calsequestrin cDNA as a template. The cDNA was excised from the bacteriophage DNA with NotI and subcloned into the pBluescript SK(−) vector (Stratagene), and DNA sequence analysis was performed by an automated DNA sequencer (Model 373A, Perkin-Elmer Applied Biosystems).Generation of Transgenic MiceA 2180-bp fragment encompassing 60 bp of 5′-untranslated region, the entire calsequestrin coding region, and 863 bp of 3′-untranslated region was generated using polymerase chain reaction methodology (flanked with SalI and EcoRV sites). This fragment was linked to the 3′-end of the mouse α-myosin heavy chain promoter and to the blunted 5′-end of the human growth hormone polyadenylation signal sequence (19Gulick J. Hewett T.E. Klevitsky R. Buck S.H. Moss R.L. Robbins J. Circ. Res. 1997; 80: 655-664Crossref PubMed Scopus (80) Google Scholar) in pBluescript SK(+). The entire 8.3-kilobase NotI fragment was excised from the plasmid sequence and used for microinjection of fertilized mouse eggs (FVB/N). To identify transgenic mice, polymerase chain reaction analysis of tail genomic DNA was carried out using a forward primer corresponding to the 5′-end of the mouse α-myosin heavy chain gene sequence (5′-CACATAGAAGCCTAGCCCACAC-3′) and a reverse primer corresponding to the 5′-end of the mouse cardiac calsequestrin cDNA sequence (5′-TTCTTCTCAGAAAGGCTGACC-3′).Morphological StudiesHistological evaluation of the hearts was performed as described previously (18Chu G. Luo W. Slack J.P. Tilgman C. Sweet W.E. Spindle M. Saupe K.W. Boivin G.P. Moravec C.S. Matlib M.A. Grupp I.L. Ingwall J.S. Kranias E.G. Circ. Res. 1996; 79: 1064-1076Crossref PubMed Scopus (110) Google Scholar). For immunocytochemistry, longitudinal sections of cardiac ventricles (20Lewis Carl S.A. Felix K. Caswell A.H. Brandt N.R. Ball Jr., W.J. Vaghy P.L. Meissner G. Ferguson D.G. J. Cell Biol. 1995; 129: 672-682Google Scholar) were fixed onto glass slides using 4% paraformaldehyde and permeabilized with phosphate-buffered saline (pH 7.4) containing 0.1% Triton X-100. After equilibration in phosphate-buffered saline (pH 7.2) containing 1% bovine serum albumin, specimens were incubated with anti-cardiac calsequestrin antibody followed by fluorescein-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). In control experiments, sections were stained with only fluorescein-conjugated anti-rabbit IgG. Sections were mounted with 100% glycerol containing 0.0025% p-phenylenediamine, 0.25% 1,4-diazabicyclo[2,2,2]octane, and 5% n-propyl gallate and examined using a Zeiss LSM 410 confocal microscope.Dot-blot AnalysisDot-blot analysis of total RNA from cardiac ventricles was performed as described previously (21D'Angelo D.D. Sakata Y. Lorenz J.N. Boivin G.P. Walsh R.A. Liggett S.B. Dorn G.W., II Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8121-8126Crossref PubMed Scopus (535) Google Scholar).Quantitative ImmunoblottingMouse hearts were homogenized at 4 °C in a buffer containing 10 mm imidazole (pH 7.0), 300 mm sucrose, 1 mm dithiothreitol, 1 mm sodium metabisulfite, 0.3 mmphenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, 2 mmEDTA, 10 μg/ml soybean trypsin inhibitor type II-S (Sigma), and 7 μg/ml pepstatin A. After solubilization of the homogenates, SDS-polyacrylamide gel electrophoresis and immunoblotting were performed as described previously (18Chu G. Luo W. Slack J.P. Tilgman C. Sweet W.E. Spindle M. Saupe K.W. Boivin G.P. Moravec C.S. Matlib M.A. Grupp I.L. Ingwall J.S. Kranias E.G. Circ. Res. 1996; 79: 1064-1076Crossref PubMed Scopus (110) Google Scholar). For the quantitation of phospholamban, the solubilized samples were boiled for 5 min to fully dissociate the pentameric form of phospholamban into monomers. Binding of the primary antibody was detected by peroxidase-conjugated secondary antibodies and ECL (Amersham Pharmacia Biotech). Protein concentration was determined by the Bradford method (Bio-Rad) with bovine serum albumin as a standard.Isolated CardiomyocytesLeft ventricular tissue was enzymatically digested to isolate individual myocytes, and measurements of myocyte mechanics and Ca2+ transients were performed at a pacing rate of 0.25 Hz as described previously (22Chu G. Dorn G.W., II Luo W. Harrer J.M. Kadambi V.J. Walsh R.A. Kranias E.G. Circ. Res. 1997; 81: 485-492Crossref PubMed Scopus (41) Google Scholar). Viability of myocytes from wild-type and transgenic mice after the isolation was ∼80 and 40%, respectively. Rod-shaped viable cells were selected for the experiments. To monitor intracellular free Ca2+transients, the cells were loaded with 7 μm Fura-2/AM, and fluorescent signals were measured (22Chu G. Dorn G.W., II Luo W. Harrer J.M. Kadambi V.J. Walsh R.A. Kranias E.G. Circ. Res. 1997; 81: 485-492Crossref PubMed Scopus (41) Google Scholar). Subsequently, the cells were perfused for 1 min with 0 Na+/0 Ca2+buffer containing 132 mm LiCl, 4.8 mm KCl, 1.2 mm MgCl2, 5 mm glucose, 10 mm HEPES, and 10 mm EGTA (pH 7.3). Then, Ca2+ transients induced by caffeine (10 mm) were obtained in the absence of Na+ and Ca2+ to determine the SR Ca2+ capacity in myocytes (23Balaguru D. Haddock P.S. Puglisi J.L. Bers D.M. Coetzee W.A. Artman M. J. Mol. Cell. Cardiol. 1997; 29: 2747-2757Abstract Full Text PDF PubMed Scopus (43) Google Scholar).ElectrophysiologyWhole-cell L-type Ca2+currents were recorded by applying depolarizing pulses every 10 s from a holding potential of −50 mV. Cell membrane capacitance was measured using voltage ramps of 0.8 V/s from a holding potential of −50 mV (24Masaki H. Sato Y. Luo W. Kranias E.G. Yatani A. Am. J. Physiol. 1997; 272: H606-H612PubMed Google Scholar). Na+-Ca2+ exchanger currents were recorded by the method of Kimura et al. (25Kimura J. Miyamae S. Noma A. J. Physiol. (Lond .). 1987; 384: 199-222Crossref Scopus (498) Google Scholar).Langendorff PerfusionContractile parameters of isolated hearts were determined at 37 °C as described previously (26Luo 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).In Vivo Left Ventricular FunctionLeft ventricular contractile parameters were assessed in closed-chest anesthetized mice using a 1.4-French scale Millar catheter (27Hoit B.D. Ball N. Walsh R.A. Am. J. Physiol. 1997; 273: H2528-H2533PubMed Google Scholar). The hearts were paced at 300 beats/min, using a 1-French scale bipolar pacing wire advanced into the right atrium through the jugular vein.MaterialsGenerous gifts of materials included rabbit cardiac calsequestrin cDNA from Dr. M. Periasamy (University of Cincinnati), mouse α-myosin heavy chain promoter from Dr. J. Robbins (Children's Hospital Medical Center, Cincinnati, OH), and rabbit polyclonal anti-junctin affinity-purified antibody (28Jones L.R. Zhang L. Sanborn K. Jorgensen A.O. Kelley J. J. Biol. Chem. 1995; 270: 30787-30796Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar) from Dr. L. R. Jones (Indiana University, Indianapolis, IN). Rabbit polyclonal anti-calsequestrin antibody was from SWant (Bellinzona, Switzerland). Monoclonal anti-triadin antibody (MA3-927), polyclonal anti-FKBP12 antibody (PA1-026), and the other primary antibodies were from Affinity BioReagents (Golden, CO).StatisticsData are presented as mean ± S.E. Comparisons between groups were evaluated using Student's ttest, with significance imparted at the p < 0.05 level.DISCUSSIONThis study demonstrated that overexpression of calsequestrin in the heart resulted in increases in SR Ca2+ storage capacity, but this pool of Ca2+ was not accessible for release, leading to attenuation of the Ca2+ transient and contractile parameters. This is the first evidence indicating that alterations in calsequestrin expression levels are associated with alterations in basal cardiac contractile parameters assessed at the cellular, organ, and intact animal levels.Cardiac-specific overexpression of calsequestrin was achieved using the α-myosin heavy chain promoter (38Subramaniam A. Jones W.K. Gulick J. Wert S. Neumann J. Robbins J. J. Biol. Chem. 1991; 266: 24613-24620Abstract Full Text PDF PubMed Google Scholar) and cDNA encoding mouse cardiac calsequestrin to assure complete homology between the overexpressed and endogenous proteins. Calsequestrin overexpression (20-fold) resulted in mild left ventricular hypertrophy without substantial morphological abnormalities. Immunofluorescence labeling of calsequestrin in transgenic ventricular tissue was generally observed as transverse striations, indicating that the overexpressed protein was predominantly localized to terminal cisternae at the Z-lines, similar to endogenous calsequestrin. Thus, the SR lumen, unlike the SR membrane (39He H. Giordano F.J. Hilal-Dandan R. Choi D.J. Rockman H.A. McDonough P.M. Bluhm W.F. Meyer M. Sayen M.R. Swanson E. Dillmann W.H. J. Clin. Invest. 1997; 100: 380-389Crossref PubMed Scopus (275) Google Scholar, 40Kadambi V.J. Ponniah S. Harrer J.M. Hoit B.D. Dorn G.W., II Walsh R.A. Kranias E.G. J. Clin. Invest. 1996; 97: 533-539Crossref PubMed Scopus (275) Google Scholar), appears capable of accommodating relatively high levels of calsequestrin expression without significant architectural alterations in cell morphology. However, these findings differ from recent observations in a transgenic mouse model overexpressing lower levels (10-fold) of dog cardiac calsequestrin in the heart (41Jones L.R. Suzuki Y.J. Wang W. Kobayashi Y.M. Ramesh V. Franzini-Armstrong C. Cleeman L. Morad M. J. Clin. Invest. 1998; 101: 1385-1393Crossref PubMed Scopus (246) Google Scholar). Introduction of the heterologous protein also resulted in cardiac hypertrophy, but there was elimination of clear striations in ventricular myocytes, suggesting disruption of normal ultrastructure.The hypertrophic response of our cardiac-specific calsequestrin-overexpressing mice was associated with induction of re-expression of a fetal gene program in the heart, particularly increases in atrial natriuretic factor, α-skeletal actin, and β-MHC transcripts. The increases in the expression of the slow MHC isoform (β-MHC) may be a contributing factor to the attenuation of contraction and relaxation rates in transgenic hearts. This depression in cardiac mechanics reflected a decrease in the amplitude of the Ca2+ transient, although the SR Ca2+ load was significantly increased as revealed by caffeine application. The apparent impairment of SR Ca2+ release in transgenic hearts may be due to (a) increased SR Ca2+-buffering capacity leading to lower intraluminal free Ca2+concentrations, which is associated with lower amounts of Ca2+ released (12Sitsapesan R. Williams A.J. J. Membr. Biol. 1997; 159: 179-185Crossref PubMed Scopus (115) Google Scholar); (b) reduced Ca2+influx through L-type Ca2+ channels, due to their lower density, leading to a smaller activation of the Ca2+-induced SR Ca2+ release (the reduced SR Ca2+ release was also evidenced by the slow inactivation kinetics of L-type Ca2+ currents, which are regulated by the transient increase in the Ca2+ concentration of the microdomain between the L-type Ca2+ channel and the ryanodine receptor (24Masaki H. Sato Y. Luo W. Kranias E.G. Yatani A. Am. J. Physiol. 1997; 272: H606-H612PubMed Google Scholar, 42Sham J.S. Cleemann L. Morad M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 121-125Crossref PubMed Scopus (206) Google Scholar)); (c) increased expression of calreticulin, which may also contribute to increased SR Ca2+ buffering and thus attenuation of the SR Ca2+ release mechanism; and (d) a defect in the coupling between L-type Ca2+ channels and ryanodine receptors in the hypertrophic transgenic cardiomyocytes, leading to a reduction in the ability of the L-type Ca2+ current to trigger SR Ca2+ release. Recently, defective excitation-contraction coupling without any changes in the densities of the L-type Ca2+ current and ryanodine receptors was observed in a rat model of cardiac hypertrophy, and this was proposed to contribute to decreases in Ca2+ transient amplitude (43Gómez A.M. Valdivia H.H. Cheng H. Lederer M.R. Santana L.F. Cannell M.B. McCune S.A. Altschuld R.A. Lederer W.J. Science. 1997; 276: 800-806Crossref PubMed Scopus (641) Google Scholar). In the calsequestrin-overexpressing hearts, there were no significant alterations in the protein levels of ryanodine receptors, but the density of the L-type Ca2+ current was decreased, similar to previous observations in models of severe pressure-overload hypertrophy or cardiac infarction (44Nuss H.B. Houser S.R. J. Mol. Cell. Cardiol. 1991; 23: 717-726Abstract Full Text PDF PubMed Scopus (70) Google Scholar, 45Santos P.E. Barcellos L.C. Mill J.G. Masuda M.O. J. Cardiovasc. Electrophysiol. 1995; 6: 1004-1014Crossref PubMed Scopus (42) Google Scholar, 46Ming Z. Nordin C. Siri F. Aronson R.S. J. Mol. Cell. Cardiol. 1994; 26: 1133-1143Abstract Full Text PDF PubMed Scopus (36) Google Scholar). Furthermore, examination of the levels of junctin and an ∼95-kDa triadin isoform, which have been proposed to act as calsequestrin-“anchoring proteins” and to form a functional complex with the ryanodine receptor (14Zhang L. Kelley J. Schmeisser G. Kobayashi Y.M. Jones L.R. J. Biol. Chem. 1997; 272: 23389-23397Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar, 35Guo W. Jorgensen A.O. Jones L.R. Campbell K.P. J. Biol. Chem. 1996; 271: 458-465Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), revealed no alterations. However, the levels of FKBP12 were significantly reduced in transgenic hearts, suggesting an important compensatory mechanism in an attempt to enhance the opening probability of the ryanodine receptors and to facilitate SR Ca2+ release, which may increase the amplitude of the cytosolic Ca2+ signal and contractility (39He H. Giordano F.J. Hilal-Dandan R. Choi D.J. Rockman H.A. McDonough P.M. Bluhm W.F. Meyer M. Sayen M.R. Swanson E. Dillmann W.H. J. Clin. Invest. 1997; 100: 380-389Crossref PubMed Scopus (275) Google Scholar). The increased open probability of ryanodine receptors may be associated with activation of calcineurin and dephosphorylation of the transcription factor NF-AT3, leading to reprogramming of gene expression and hypertrophy (47Molkentin J.D. Lu J.R. Antos C.L. Markham B. Richardson J. Robbins J. Grant S.R. Olson E.N. Cell. 1998; 93: 215-228Abstract Full Text Full Text PDF PubMed Scopus (2191) Google Scholar). Furthermore, down-regulation of FKBP12, which has been postulated to interact with and to inhibit the activity of transforming growth factor-β signaling (48Wang T. Li B.-Y. Danielson P.D. Shah P.C. Rockwell S. Lechleider R.J. Martin J. Manganaro T. Donahoe P.K. Cell. 1996; 86: 435-444Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar), may also contribute to development of cardiac hypertrophy through the transforming growth factor-β pathway. Treatment of the calsequestrin-overexpressing mice with cyclosporin (47Molkentin J.D. Lu J.R. Antos C.L. Markham B. Richardson J. Robbins J. Grant S.R. Olson E.N. Cell. 1998; 93: 215-228Abstract Full Text Full Text PDF PubMed Scopus (2191) Google Scholar) may allow us to distinguish between these pathways for the hypertrophic response in this model.Assessment of the protein levels of the SR Ca2+-ATPase and its regulator phospholamban indicated that calsequestrin overexpression was associated with increases compared with wild-type littermates. It is interesting to note that the increases in the levels of these proteins were similar, suggesting that the maximal velocity of the SR Ca2+-ATPase activity was increased, without any changes in the affinity of this enzyme for Ca2+ (49Harrer J.M. Haghighi K. Kim H.W. Ferguson D.G. Kranias E.G. Am. J. Physiol. 1997; 272: H57-H66PubMed Google Scholar). The increased maximal velocity of the SR Ca2+ pump probably constitutes an important compensatory response in an attempt to increase the amplitude of the Ca2+ transient in transgenic cardiomyocytes. Decreases in contractile parameters were also observed in intact hearts and in closed-chest animals. Reduction of left ventricular isovolumic parameters indicated that calsequestrin overexpression was uncompensated in vivo. Thus, the development of hypertrophy may provide an adaptive response to normalize contractile dysfunction in this animal model. However, induction of the fetal gene program and other compensatory responses, accompanying calsequestrin overexpression, make it rather difficult to assess the direct effects of altered calsequestrin levels on cardiac contractile parameters.In summary, our findings indicate that calsequestrin is a regulator of the cardiac SR Ca2+ storage capacity and the amount of Ca2+ available for release during excitation-contraction coupling. Thus, alterations in the expression levels of this protein would reflect alterations in the levels of cytosolic Ca2+and contractile parameters. The availability of transgenic models with altered expression of calsequestrin will facilitate further studies on elucidating the mechanism(s) by which calsequestrin buffers SR luminal Ca2+, the effect(s) of free luminal Ca2+ on SR Ca2+ release and ryanodine receptor gating properties, and the dynamic relationship between changes in SR luminal Ca2+and cytosolic Ca2+ levels during the contractile cycle. Such studies will greatly improve our understanding of the regulatory mechanisms involved in SR Ca2+ cycling and contractility in the mammalian myocardium. In cardiac excitation-contraction coupling, the sarcoplasmic reticulum (SR)1 plays an essential role in the regulation of the cytosolic free Ca2+concentration. There are three major functions of the SR: (a) Ca2+ uptake from the cytosol into the SR lumen, resulting in muscle relaxation; (b) Ca2+storage in the SR lumen; and (c) Ca2+ release from the SR into the cytosol, resulting in muscle contraction. The main SR proteins responsible for these functions are the Ca2+transport ATPase, the Ca2+ storage protein calsequestrin (1Yano K. Zarain-Herzberg A. Mol. Cell. Biochem. 1994; 135: 61-70Crossref PubMed Scopus (137) Google Scholar), and the Ca2+ release channel or ryanodine receptor, respectively. Phospholamban is another SR protein that plays a crucial role in the modulation of myocardial contractility and relaxation (2Koss K.L. Kranias E.G. Circ. Res. 1996; 79: 1059-1063Crossref PubMed Scopus (288) Google Scholar). Recent studies with isolated human myocardial preparations suggested that the impaired Ca2+ handling of the SR may be an important subcellular mechanism contributing to the depressed contractility in heart failure (3Hasenfuss G. Reinecke H. Studer R. Pieske B. Meyer M. Drexler H. Just H. Basic Res. Cardiol. 1996; 91 Suppl. 2: 17-22Crossref PubMed Google Scholar, 4Wankerl M. Schwartz K. J. Mol. Med. 1995; 73: 487-496Crossref PubMed Scopus (55) Google Scholar, 5Beuckelmann D.J. Nabauer M. Erdmann E. Circulation. 1992; 85: 1046-1055Crossref PubMed Scopus (720) Google Scholar). However, it is still controversial whether the expression levels of SR Ca2+-handling proteins are altered in failing human hearts. Some groups have reported alterations in mRNA levels and/or protein expression levels of SR Ca2+-ATPase, phospholamban, and the SR Ca2+ release channel in human heart failure (3Hasenfuss G. Reinecke H. Studer R. Pieske B. Meyer M. Drexler H. Just H. Basic Res. Cardiol. 1996; 91 Suppl. 2: 17-22Crossref PubMed Google Scholar, 6Hasenfuss G. Meyer M. Schillinger W. Preuss M. Pieske B. Just H. Basic Res. Cardiol. 1997; 92 Suppl. 1: 87-93Crossref PubMed Google Scholar, 7Arai M. Alpert N.R. MacLennan D.H. Barton P. Periasamy M. Circ. Res. 1993; 72: 463-469Crossref PubMed Scopus (433) Google Scholar). However, other groups observed no significant changes in the expression levels of these proteins in hearts with end-stage heart failure (8Movsesian M.A. Karimi M. Green K. Jo
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