Targeted Inhibition of Ca2+/Calmodulin-dependent Protein Kinase II in Cardiac Longitudinal Sarcoplasmic Reticulum Results in Decreased Phospholamban Phosphorylation at Threonine 17
2003; Elsevier BV; Volume: 278; Issue: 27 Linguagem: Inglês
10.1074/jbc.m302193200
ISSN1083-351X
AutoresYong Ji, Bailing Li, Thomas D. Reed, John N. Lorenz, Marcia A. Kaetzel, John Dedman,
Tópico(s)Cardiomyopathy and Myosin Studies
ResumoTo investigate the role of Ca2+/calmodulin-dependent kinase II in cardiac sarcoplasmic reticulum function, transgenic mice were designed and generated to target the expression of a Ca2+/calmodulin-dependent kinase II inhibitory peptide in cardiac longitudinal sarcoplasmic reticulum using a truncated phospholamban transmembrane domain. The expressed inhibitory peptide was highly concentrated in cardiac sarcoplasmic reticulum. This resulted in a 59.7 and 73.6% decrease in phospholamban phosphorylation at threonine 17 under basal and β-adrenergic stimulated conditions without changing phospholamban phosphorylation at serine 16. Sarcoplasmic reticulum Ca2+ uptake assays showed that the Vmax was decreased by ∼30% although the apparent affinity for Ca2+ was unchanged in heterozygous hearts. The in vivo measurement of cardiac function showed no significant reductions in positive and negative dP/dt, but a moderate 18% decrease in dP/dt40, indicative of isovolumic contractility, and a 26.1% increase in the time constant of relaxation (τ) under basal conditions. The changes in these parameters indicate a moderate cardiac dysfunction in transgenic mice. Although the 3and 4-month-old transgenic mice displayed no overt signs of cardiac disease, when stressed by gestation and parturition, the 7-month-old female mice develop dilated heart failure, suggesting the important role of Ca2+/calmodulin-dependent kinase II pathway in the development of cardiac disease. To investigate the role of Ca2+/calmodulin-dependent kinase II in cardiac sarcoplasmic reticulum function, transgenic mice were designed and generated to target the expression of a Ca2+/calmodulin-dependent kinase II inhibitory peptide in cardiac longitudinal sarcoplasmic reticulum using a truncated phospholamban transmembrane domain. The expressed inhibitory peptide was highly concentrated in cardiac sarcoplasmic reticulum. This resulted in a 59.7 and 73.6% decrease in phospholamban phosphorylation at threonine 17 under basal and β-adrenergic stimulated conditions without changing phospholamban phosphorylation at serine 16. Sarcoplasmic reticulum Ca2+ uptake assays showed that the Vmax was decreased by ∼30% although the apparent affinity for Ca2+ was unchanged in heterozygous hearts. The in vivo measurement of cardiac function showed no significant reductions in positive and negative dP/dt, but a moderate 18% decrease in dP/dt40, indicative of isovolumic contractility, and a 26.1% increase in the time constant of relaxation (τ) under basal conditions. The changes in these parameters indicate a moderate cardiac dysfunction in transgenic mice. Although the 3and 4-month-old transgenic mice displayed no overt signs of cardiac disease, when stressed by gestation and parturition, the 7-month-old female mice develop dilated heart failure, suggesting the important role of Ca2+/calmodulin-dependent kinase II pathway in the development of cardiac disease. Calcium plays a central role in cardiac excitation-contraction coupling. The sarcoplasmic reticulum (SR) 1The abbreviations used are: SR, sarcoplasmic reticulum; CaMKII, Ca2+/calmodulin-dependent kinase II; SERCA, SR Ca2+-ATPase; TG, transgenic; NTG, non-transgenic; PLB, phospholamban; PKA, cAMP-dependent protein kinase; AIP, autocamitide inhibitory peptide; α-MHC, α-myosin heavy chain; RyR, ryanodine receptor; LV, left ventricle; LVEDP, LV end-diastolic pressure; PP, protein phosphatases. 1The abbreviations used are: SR, sarcoplasmic reticulum; CaMKII, Ca2+/calmodulin-dependent kinase II; SERCA, SR Ca2+-ATPase; TG, transgenic; NTG, non-transgenic; PLB, phospholamban; PKA, cAMP-dependent protein kinase; AIP, autocamitide inhibitory peptide; α-MHC, α-myosin heavy chain; RyR, ryanodine receptor; LV, left ventricle; LVEDP, LV end-diastolic pressure; PP, protein phosphatases. releases Ca2+ to trigger contraction and uptakes Ca2+ to initiate relaxation. Ca2+-induced Ca2+ release occurs via ryanodine-sensitive SR Ca2+ release channels located mainly at junctional SR ("foot" structure), whereas Ca2+ uptake is mediated principally by the SR Ca2+-ATPase (SERCA) which is located in the longitudinal SR. Both Ca2+ release and uptake are proposed to be regulated in a Ca2+-dependent manner via Ca2+/calmodulin-dependent protein kinase II (CaMKII) (1Maier L.S. Bers D.M. J. Mol. Cell. Cardiol. 2002; 34: 919-939Google Scholar). CaMKII is a member of a family of Ca2+/calmodulin-regulated enzymes. Four CaMKII isoforms are derived from four closely related genes, α, β, γ, and δ (2Schuman H. Curr. Opin. Cell Biol. 1993; 5: 247-253Google Scholar). The δ and γ are the primary cardiac CaMKII isoforms expressed in the adult heart (3Tobimatsu T. Fujisawa H. J. Biol. Chem. 1989; 264: 17907-17912Google Scholar, 4Mayer P. Mohlig M. Idlibe D. Pfeiffer A. Basic Res. Cardiol. 1995; 90: 372-379Google Scholar, 5Hagemann D. Hoch B. Krause E.G. Karczewski P. J. Cell. Biochem. 1999; 74: 202-210Google Scholar). CaMKII distributes in distinct compartments of the cardiomyocytes including sarcolemma, cytosol, SR, and nucleus (6Braun A.P. Schulman H. Annu. Rev. Physiol. 1995; 57: 417-445Google Scholar), which may represent its functional relevance in the heart. The link between specific isoforms of CaMKII with particular regulatory properties, intracellular localization, and cellular substrates is not established. The activity of SERCA is primarily regulated by an SR intrinsic protein, phospholamban (PLB). PLB physically interacts with SERCA to inhibit pump activity (7Tada M. Kirchberger M.A. Repke D.L. Katz A.M. J. Biol. Chem. 1974; 249: 6174-6180Google Scholar). The phosphorylation of PLB disrupts the interaction of PLB with the SERCA pump, relieving its inhibitory effect and resulting in an increase in the apparent affinity of SERCA for Ca2+ (7Tada M. Kirchberger M.A. Repke D.L. Katz A.M. J. Biol. Chem. 1974; 249: 6174-6180Google Scholar). cAMP-dependent protein kinase (PKA) mediates PLB phosphorylation at serine 16 (7Tada M. Kirchberger M.A. Repke D.L. Katz A.M. J. Biol. Chem. 1974; 249: 6174-6180Google Scholar, 8Simmerman H.K.B. Collins J.H. Theibert J.L. Wegener A.D. Jones L.R. J. Biol. Chem. 1986; 258: 13587-13591Google Scholar), whereas CaMKII mediates phosphorylation of PLB at threonine 17 (8Simmerman H.K.B. Collins J.H. Theibert J.L. Wegener A.D. Jones L.R. J. Biol. Chem. 1986; 258: 13587-13591Google Scholar, 9Lepeuch C.J. Haiech J. Demaille J.G. Biochemistry. 1979; 18: 5150-5157Google Scholar). In the intact heart, both PLB serine 16 and threonine 17 are phosphorylated by PKA and CaMKII, respectively, in response to β-adrenergic stimulation (10Wegener A.D. Simmermann H.K.B. Lindemann J.P. Jones L.R. J. Biol. Chem. 1989; 264: 11469-11474Google Scholar, 11Talosi L. Edes I. Kranias E.G. Am. J. Physiol. 1993; 264: H791-H797Google Scholar). In vitro studies have indicated that SERCA is also phosphorylated by CaMKII at Ser38, which may enhance the maximal velocity (Vmax) of calcium uptake (12Hawkins C. Xu A. Narayanan N. J. Biol. Chem. 1994; 269: 31198-31206Google Scholar, 13Toyofuku T. Kurzydloski K. Narayanan N. MacLennan D.H. J. Biol. Chem. 1994; 269: 26492-26496Google Scholar, 14Xu A. Narayanan N. J. Biol. Chem. 2000; 275: 4407-4416Google Scholar). However, this pathway remains controversial (15Reddy L.G. Jones L.R. Pace R.C. Stockes D.L. J. Biol. Chem. 1996; 271: 14964-14970Google Scholar, 16Odermatt A. Kazimieez K. MacLennan D.H. J. Biol. Chem. 1996; 271: 14206-14213Google Scholar), due to a lack of in vivo evidence. In addition to Ca2+ uptake, CaMKII may also play a role in regulating Ca2+-induced Ca2+ release by phosphorylation of serine 2809 of the cardiac SR calcium release channel (ryanodine receptor, RyR) which can also be a target for PKA (17Witcher D.R. Kovacs R.J. Schulman H. Cefali D.C. Jones L.R. J. Biol. Chem. 1991; 266: 11144-11152Google Scholar, 18Hain J. Onoue H. Mayrleitner M. Fleischer S. Schindler H. J. Biol. Chem. 1995; 270: 2074-2081Google Scholar). However, whether the phosphorylation of RyR leads to opening or closing the Ca2+ release channel remains undefined (17Witcher D.R. Kovacs R.J. Schulman H. Cefali D.C. Jones L.R. J. Biol. Chem. 1991; 266: 11144-11152Google Scholar, 18Hain J. Onoue H. Mayrleitner M. Fleischer S. Schindler H. J. Biol. Chem. 1995; 270: 2074-2081Google Scholar, 19Lokuta A.J. Roger T.B. Lederer W.J. Valdivia H.H. J. Physiol. (Lond.). 1995; 487: 609-622Google Scholar). Therefore, the role of CaMKII in the regulation of RyR in intact heart is not clear. Recently, several studies (20Hoch B. Meyer R. Hetzer R. Krause E.G. Karczewski P. Circ. Res. 1999; 84: 713-721Google Scholar) have shown that the level of cardiac isoform CaMKII δ3 is significantly increased in dilated cardiomyopathy patients. Currie (21Currie S. Smith G.L. FEBS Lett. 1999; 459: 244-248Google Scholar) and Kirchhefer et al. (22Kirchhefer U. Schmitz W. Scholz H. Neumann J. Cardiovasc. Res. 1999; 42: 254-261Google Scholar) have independently reported that the activity of CaMKII is significantly increased in hypertrophied animal models as well as in human heart failure. However, Netticadan et al. (23Netticadan T. Temsah R. Osada M. Dhallar N.S. Am. J. Physiol. 1999; 277: C384-C391Google Scholar, 24Netticadan T. Temsah R. Kawabata K. Dhalla N.S. Circ. Res. 2000; 86: 596-605Google Scholar) showed that the endogenous SR-associated CaMKII-mediated phosphorylation of SR Ca2+-handling proteins is depressed in heart failure due to either ischemia-reperfusion or myocardial infarction. These data, taken together, suggest that SR CaMKII is associated with the abnormal Ca2+ handling of the SR in cardiomyocytes. However, the role of CaMKII in the regulation of SR Ca2+-handling proteins under pathophysiological conditions is not clear. In order to investigate the role of SR-associated CaMKII in the regulation of SR Ca2+ transport, as well as in regulating cardiac function, transgenic mice were generated to target specifically the expression of CaMKII inhibitory peptide to cardiac longitudinal SR. The SR targeting sequence is defined by the transmembrane portion of the PLB protein, encoding a double mutant that obviates PLB function. We demonstrate that targeted inhibition of SR CaMKII activity results in a significant decrease in PLB phosphorylation at threonine 17. When stressed, the transgenic mice develop dilated heart failure, suggesting the important role of CaMKII pathway in the development of cardiac disease. Generation of CaMKII-AIP4Transgenic Mice—A synthetic gene expression unit was engineered to encode three functional domains using oligonucleotides produced by the DNA Core Facility at the University of Cincinnati. Starting at the amino terminus, the expression unit contains nucleotides that will encode a CaMKII inhibitor, a FLAG epitope, and an SR localization signal. The CaMKII inhibitor domain consisted of sequences encoding a tetramer of the CaMKII autocamitide inhibitory peptide (AIP4), this 13-amino acid sequence (KKALRRQEAVDAL) is known to be a highly specific and potent inhibitor of CaMKII (25Ishida A. Kameshita I. Okuno S. Kitani T. Fujisawa H. Biochem. Biophys. Res. Commun. 1995; 212: 806-812Google Scholar). Sequences encoding a FLAG epitope (amino acids DYKDDDDK) (Eastman Kodak Co.) were placed 3′ of the AIP tetramer. In addition, a synthetic gene encoding a truncated PLB transmembrane domain (amino acids 23–52) was inserted to serve as SR localization signal with mutations at Leu31 (L31A) and Asn34 (N34A) (Genomatix, Ltd., patent pending). Previous studies have shown that mutants L31A and N34A result in loss of the PLB inhibition to SERCA activity (26Kimura Y. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 1997; 272: 15061-15064Google Scholar). This expression unit was subsequently subcloned into a pBluescript-based vector between the 5.5-kb murine α-myosin heavy chain (α-MHC) promoter (a gift from J. Robbins, Children's hospital, Cincinnati, OH) and an SV40 polyadenylation signal. The linear transgene fragments were released from SalI sites and were purified by gel purification kit (Qiagen). The transgene was injected into pronuclei of fertilized mouse oocytes by the Transgenic Core at University of Cincinnati. The resultant pups were screened for the presence of the transgene by PCR, using an α-MHC-specific primer (5′-GCCCACACCAGAAATGACAGA-3′) and an AIP-specific primer (5′-ACTCGAGCAGGAGCATGACGATA-3′). The founder mice were confirmed by Southern blot analyses. Founder mice were bred with FVB/N wild type mice. Heterozygous animals from at least the third generation were used for all studies, with their non-transgenic (NTG) littermates serving as controls. All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee. Southern Blotting Analysis—Southern blotting analysis was performed to determine the copy number. According to the formula, 1 copy of transgene = 6 × 109 bp (genomic DNA/copy) × μg of transgene DNA loaded/bp of transgene, different amounts of transgene DNA was loaded to estimate the different copy number that served as control. Ten μg of genomic DNA prepared from tail biopsies was digested with KpnI and electrophoresed. The blots were hybridized with the SalI to SalI fragment of the released transgene. The TG samples produce 5.8and 1-kb bands. Intensities of the TG band were measured using a PhosphorImager (Amersham Biosciences) and were compared with the control bands to estimate copy number. Preparation of Cardiac Homogenates, SR-enriched Microsomes, and Cytosol Fraction—Cardiac homogenates, SR-enriched membrane fractions, as well as cytosol fractions were prepared as described previously with a slight modification (27Ji Y. Loukianov E. Periasamy M. Anal. Biochem. 1999; 269: 236-244Google Scholar). In brief, an individual mouse heart was used for preparation of cardiac homogenates with 1 ml of ice-cold buffer A containing (mm) 10 imidazole, pH 7.0, 300 sucrose, 10 NaF, 1 EDTA, 0.3 phenylmethylsulfonyl fluoride, 0.5 dithiothreitol, and proteinase inhibitors. For SR isolation, 10–15 pooled mouse hearts were homogenized. Cardiac homogenates were centrifuged at 8,000 × g for 20 min; the supernatant was collected, and the resulting pellet (first pellet) was homogenized in buffer A and re-centrifuged as above. The supernatant from two spins were pooled, mixed with buffer B (same as buffer A except that 300 mm sucrose was substituted by NaCl to obtain the final concentration of 600 mm), and centrifuged at 100,000 × g for 60 min. The supernatant was concentrated through Centriprep (Millipore) and was treated as a cytosol fraction. The resulting pellet (second pellet) was washed in buffer A and re-centrifuged at 100,000 × g for an additional 60 min. The final pellet was used as an SR membrane. Protein concentration was measured by Bradford assay (Bio-Rad). Quantitative Immunoblotting Analysis—The antibodies used for immunoblotting were as follows: mouse anti-FLAG M2 and anti-PP1 α-subunit antibodies (Sigma), polyclonal anti-calsequestrin and monoclonal anti-PLB, anti-RyR antibodies (Affinity Bioreagents, Inc.), mouse anti-PP2A antibody and rabbit anti-CaMKII-δ antibody (Calbiochem). The appropriate secondary antibodies, horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG, were obtained from Sigma. Enhanced chemiluminescence was performed using the Super-Signal Chemiluminescent Detection System (Amersham Biosciences). Calsequestrin protein was used as internal control. The data were analyzed using Image Pro 4.0 (Media Cybernetics). Immunocytochemical Staining—Whole hearts were taken from NTG and TG mice. Tissue-Tek O.C.T compound was added to merge the hearts into a chamber, and then the hearts were immediately frozen in liquid N2. Frozen tissue sections (4 μm) were stained as described previously (28Wang Y. Takashi E. Xu M. Ayub A. Ashraf M. Circulation. 2001; 104: 85-90Google Scholar). The FLAG expression was detected using mouse anti-FLAG antibody (1:10 dilution) followed by fluorescein isothiocyanate-conjugated goat anti-mouse IgG antibody (Cappel, 1:200 dilution). The same tissue section was used to detect SERCA2a protein using polyclonal anti-SERCA2a antibody (a gift from Dr. Frank Wyutack in Katholieke Universiteit Leuven, Belgium, 1:200 dilution) followed by fluorescein Cy-3-conjugated goat anti-rabbit IgG antibody (Cappel, 1:200 dilution). The tissue sections were observed by a Nikon 135 optiphot fluorescence microscope. Immunodetection of Site-specific Phosphorylation of PLB and RyR—To determine the basal phosphorylation level of PLB and RyR, polyclonal antibodies raised against a PLB peptide phosphorylated at Ser16 (PLB-phosphoserine 16) or at Thr17 (PLB-phosphothreonine 17) (Cyclacel, Dundee, UK) and polyclonal antibody against phosphorylated RyR-2809 (Badrilla, Leeds, UK) were used. The mouse hearts were excised and immediately freeze-clamped using instruments pre-cooled with liquid N2 to ensure complete and rapid freezing of the cardiac tissues. Homogenates were prepared, and the following procedures were performed according to the methods described previously (29Ji Y. Lalli M.J. Babu G.J. Xu Y. Kirkpatrick D.L. Liu L.H. Chiamvimonvat N. Walsh R.A. Shull G.E. Periasamy M. J. Biol. Chem. 2000; 275: 38073-38080Google Scholar). In vitro phosphorylation was performed using cardiac homogenates from mouse heart, as described previously (30Luo W. Chu G. Sato Y. Zhou Z. Kadambi V.J. Kranias E.G. J. Biol. Chem. 1998; 273: 4734-4739Google Scholar). For endogenous CaMKII phosphorylation, 10 μl (25 μg) of the cardiac homogenates was added to 10 μl of reaction mixture containing 20 mm imidazole (pH 7.0), 10 mm MgCl2,10mm NaF, 0.5 mm EGTA, 0.1 mm ATP. Additionally, 0.5 mm CaCl2, 2 μm calmodulin, and 1 μm protein kinase inhibitor peptide 5–24 amide (Sigma) were included. The reaction was carried out at 30 °C. PKA phosphorylation of the cardiac homogenates was carried out in the above-described reaction mixture with 20 units of the PKA catalytic subunit. Reactions were terminated with 4 μl of 6× SDS sample buffer after a 5-(CaMKII) and 2-min (PKA) incubation, which was associated with optimal phosphate incorporation in PLB. Twenty μg of protein was subject to 15% SDS-PAGE followed by the detection of PLB Ser16 and PLB Thr17 using the appropriate phosphorylated PLB antibodies as described above. In vivo phosphorylation was carried out by perfusion with increments from 1 to 32 ng/g/min dobutamine only or followed by 50 mg/ml Ca2+ via left jugular vein. The hearts were rapidly freeze-clamped with liquid N2 and homogenized. Twenty μg of cardiac homogenate protein was used for the detection of PLB Ser16 and PLB Thr17 as described above. To detect phosphorylated RyR, 20 μg of homogenate protein was subjected to 6% SDS-PAGE followed by probing with anti-phosphorylated RyR-2809 antibody. After stripping the membrane, the same blot was used to probe with anti-RyR antibody to confirm the right location of the protein on the membrane. Ca2+Transport Assays—Oxalate-facilitated Ca2+ uptake into SR vesicles in cardiac homogenates was determined by the Millipore filtration technique following an established method (31Ji Y. Loukianov E. Loukianova T. Jones L.R. Periasamy M. Am. J. Physiol. 1999; 276: H89-H97Google Scholar). Cardiac homogenates (100 μg/ml) were incubated (37 °C) in uptake medium containing various concentrations of CaCl2 to yield 0.03–3 μm free Ca2+ (containing 1 μCi/μmol 45Ca2+), as determined by the computer program (32Robertson S. Potter J.D. Schwartz A. Methods in Pharmacology. Plenum Publishing Corp., New York1984: 63-75Google Scholar). The reaction was initiated by the addition of 5 mm ATP. The rate of Ca2+ uptake was calculated by least squares linear regression analysis of uptake at 30, 60, and 90 s. Data were analyzed by Origin 7.0 (Microcal Software). SR Protein Phosphatase Activity Assay—The SR protein phosphatase activity was determined with the Ser/Thr phosphatase assay kit from Upstate Biotechnology. The reaction was initiated by adding SR (10 μg) or cytosol (5 μg) proteins prepared as described above to microtiter wells with and without the synthetic substrate (200 μm) in a total assay volume of 25 μl for 20 min. The reaction was terminated by the addition of 100 μl of Malachite Green solution. The absorbance was read at 630 nm to determine the inorganic phosphate released. The phosphatase activity was calculated by subtracting the OD values in the absence of the substrate from those in its presence (24Netticadan T. Temsah R. Kawabata K. Dhalla N.S. Circ. Res. 2000; 86: 596-605Google Scholar). Hemodynamic Measurements—Hemodynamic function was measured as described previously (33Lorenz J.N. Robbins J. Am. J. Physiol. 1997; 272: H1137-H1146Google Scholar). Briefly, animals were anesthetized with 50 μg/g body weight ketamine and 100 μg/g body weight thiobutabarbital (Inactin, Research Biochemicals International, Natick, MA) and placed on a thermally controlled surgical table. Following tracheostomy, the right femoral artery and vein were cannulated with polyethylene tubing (OD 0.3–0.5 mm). The arterial catheter was connected to a COBE CDXIII fixed dome pressure transducer (Cobe Cardiovascular, Arvada, CO) for measurement of arterial blood pressure, and the venous catheter was connected to a syringe pump for the infusion of experimental drugs. A high fidelity, 1.4 French (0.5 mm) micromanometer (SPR-671, Millar Instruments, Houston, TX) was then inserted into the right carotid artery and advanced across the aortic valve and into the left ventricle. After completion of the surgery animals were allowed to stabilize for 30–45 min. Cardiovascular responses to separate doses of dobutamine, delivered as a constant infusion (0.1 μl/min/g body weight) over a 3-min period, were then determined; animals were allowed to recover to base line for 5–10 min between doses. After completion of the dose-response protocol, 50 mg/ml CaCl2 was infused at a rate of 0.2 μl/min/g body weight, and measurements of heart function were repeated after 5 min. To evaluate left ventricular function, the following measurements and calculations were made: the maximal positive first derivation of LV pressure (peak + dP/dt), an indicator of myocardial contractility; the rate of LV pressure increase determined at an LV pressure of 40 mm Hg (dP/dt40), an indicative of isovolumic contractility (34Shannon T.R. Ginsburg K.S. Bers D.M. Biophys. J. 2000; 78: 334-343Google Scholar, 35Tang W. Weil M.H. Sun S. Noc M. Yang L.Y. Gazmuri R.J. Circulation. 1995; 92: 3089-3093Google Scholar); the time constant of relaxation (τ), a measure of LV relaxation, calculated from the exponential decay of the LV pressure trace to a zero asymptote LV; end-diastolic pressure (LVEDP), an index of LV pre-load and compliance (36Stefanadis C. Manolis A. Dernellis J. Tsioufis C. Tsiamis E. Gavras I. Gavras H. Toutouzas P. J. Hum. Hypertens. 2001; 15: 635-642Google Scholar). Pressure signals were recorded and analyzed using a PowerLab 4/s data acquisition system (ADInstruments, Colorado Springs, CO). Average values and each variable were determined for 20–30-s periods at the end of each 3-min dose. Analytical and Statistical Procedures—Data are reported as the means ± S.E. The statistical analysis was performed using unpaired Student's t test or one factor (within) or mixed, two-factor analysis of variance (for cardiac function data). A p value of 0.05 versus NTG.View Large Image Figure ViewerDownload (PPT) The 12–16 week-old TG mice were capable of producing healthy litters that exhibited no overt manifestations of a diseased phenotype. There was no evidence of cardiac hypertrophy in the TG mice. The heart weight/body weight ratio (mg/g) of the 12–16-week-old mice used below for the analysis of hemodynamic function, PLB phosphorylation, and SR Ca2+ uptake was 5.35 ± 0.03 for NTG and 5.33 ± 0.11 for TG in line 46 (n = 10, p > 0.05). Cardiac Expression of AIP4Results in Decreased Phosphorylation of Phospholamban—PLB is regulated by phosphorylation of Thr17 by CaMKII and of Ser16 by PKA (7Tada M. Kirchberger M.A. Repke D.L. Katz A.M. J. Biol. Chem. 1974; 249: 6174-6180Google Scholar, 8Simmerman H.K.B. Collins J.H. Theibert J.L. Wegener A.D. Jones L.R. J. Biol. Chem. 1986; 258: 13587-13591Google Scholar, 9Lepeuch C.J. Haiech J. Demaille J.G. Biochemistry. 1979; 18: 5150-5157Google Scholar). To determine the effect of SR-targeted expression of AIP4 on PLB phosphorylation, the PLB phosphorylation levels under either basal or stimulated conditions were examined. As shown in Fig. 4A, the basal levels of both high and low molecular forms of Thr17-phosphorylated PLB were significantly decreased by 59.7% in extracts prepared from 14to 16-week-old TG mouse hearts (arbitrary units: 1.426 ± 0.075 in NTG versus 0.574 ± 0.094 in TG, n = 6, p < 0.01). However, Ser16-phosphorylated PLB in TG mice was not significantly altered (1.041 ± 0.024 in NTG versus 0.959 ± 0.052 in TG, n = 4, p = 0.104). Our results indicate that functional expression of AIP4 is evidenced by exclusively blocking the CaMKII pathway. Furthermore, we examined the PLB phosphorylation under in vitro stimulated conditions. Cardiac homogenates from NTG and TG hearts were incubated with calmodulin (CaM) in the presence of Ca2+ under optimal conditions and subjected to SDS-PAGE followed by Western blot using PT-17 antibody. As shown in Fig. 4B, in the absence and presence of calmodulin and Ca2+, Thr17-phosphorylated PLB, were decreased by 63.9 (1.469 ± 0.060 in NTG versus 0.530 ± 0.017 in TG, n = 3, p < 0.01) and 47.8% (1.315 ± 0.039 in NTG versus 0.686 ± 0.096 in TG, n = 3, p < 0.01), respectively, in TG hearts compared with NTG controls, indicating the endogenous CaMKII activity in TG hearts is dramatically decreased. On the other hand, PKA-dependent phosphorylation of PLB was not significantly altered as represented by the similar pattern of Ser16-phosphorylated PLB in the presence of PKA catalytic subunit between NTG (increased by 48.4%, n = 4) and TG (increased by 50.4%, n = 4) hearts (Fig. 4B). This result suggested that the PKA pathway is not affected by AIP expression on SR. It has been reported that β-adrenergic stimulation was associated with significant PLB phosphorylation at both Ser16 and Thr17 sites. To observe the effect of β-adrenergic stimulation on phosphorylation of PLB, the NTG and TG mouse hearts were perfu
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