Sorcin Regulates Excitation-Contraction Coupling in the Heart
2003; Elsevier BV; Volume: 278; Issue: 31 Linguagem: Inglês
10.1074/jbc.m302009200
ISSN1083-351X
AutoresMarian B. Meyers, Avi Fischer, Yanjie Sun, Coeli M. Lopes, Tibor Rohács, Tomoe Y. Nakamura, Yingying Zhou, Paul C. Lee, Ruth A. Altschuld, Sylvia A. McCune, William A. Coetzee, Glenn I. Fishman,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoSorcin is a penta-EF hand Ca2+-binding protein that associates with both cardiac ryanodine receptors and L-type Ca2+ channels and has been implicated in the regulation of intracellular Ca2+ cycling. To better define the function of sorcin, we characterized transgenic mice in which sorcin was overexpressed in the heart. Transgenic mice developed normally with no evidence of cardiac hypertrophy and no change in expression of other calcium regulatory proteins. In vivo hemodynamics revealed significant reductions in global indices of contraction and relaxation. Contractile abnormalities were also observed in isolated adult transgenic myocytes, along with significant depression of Ca2+ transient amplitudes. Whole cell ICa density and the time course of activation were normal in transgenic myocytes, but the rate of inactivation was significantly accelerated. These effects of sorcin on L-type Ca2+ currents were confirmed in Xenopus oocyte expression studies. Finally, we examined the expression of sorcin in normal and failing hearts from spontaneous hypertensive heart failure rats. In normal myocardium, sorcin extensively co-localized with ryanodine receptors at the Z-lines, whereas in myopathic hearts the degree of co-localization was markedly disrupted. Together, these data indicate that sorcin modulates intracellular Ca2+ cycling and Ca2+ influx pathways in the heart. Sorcin is a penta-EF hand Ca2+-binding protein that associates with both cardiac ryanodine receptors and L-type Ca2+ channels and has been implicated in the regulation of intracellular Ca2+ cycling. To better define the function of sorcin, we characterized transgenic mice in which sorcin was overexpressed in the heart. Transgenic mice developed normally with no evidence of cardiac hypertrophy and no change in expression of other calcium regulatory proteins. In vivo hemodynamics revealed significant reductions in global indices of contraction and relaxation. Contractile abnormalities were also observed in isolated adult transgenic myocytes, along with significant depression of Ca2+ transient amplitudes. Whole cell ICa density and the time course of activation were normal in transgenic myocytes, but the rate of inactivation was significantly accelerated. These effects of sorcin on L-type Ca2+ currents were confirmed in Xenopus oocyte expression studies. Finally, we examined the expression of sorcin in normal and failing hearts from spontaneous hypertensive heart failure rats. In normal myocardium, sorcin extensively co-localized with ryanodine receptors at the Z-lines, whereas in myopathic hearts the degree of co-localization was markedly disrupted. Together, these data indicate that sorcin modulates intracellular Ca2+ cycling and Ca2+ influx pathways in the heart. Cardiac contractility is dependent upon the precise regulation of intracellular Ca2+ cycling. The Ca2+ cycle is initiated by activation of voltage-operated L-type Ca2+ channels and the subsequent triggering of Ca2+ release from the sarcoplasmic reticulum via ryanodine receptors (RyRs). 1The abbreviations used are: RyR, ryanodine receptor; SERCA2, sarco/endoplasmic reticulum Ca-ATPase; SHHF, spontaneous-hypertensive heart failure; TG, transgenic; WT, wild type; PBS, phosphate-buffered saline.1The abbreviations used are: RyR, ryanodine receptor; SERCA2, sarco/endoplasmic reticulum Ca-ATPase; SHHF, spontaneous-hypertensive heart failure; TG, transgenic; WT, wild type; PBS, phosphate-buffered saline. Restoration of intracellular Ca2+ levels to diastolic levels is in turn accomplished by various efflux mechanisms involving key transport proteins such as SERCA2 and the Na+-Ca2+ exchanger (1Bers D.M. Nature. 2002; 415: 198-205Crossref PubMed Scopus (3260) Google Scholar). Additional modulatory proteins such as phospholamban, FKBP12, and calsequestrin participate in these functions, and our previous studies suggested that sorcin may also serve as a modulatory or accessory protein in intracellular Ca2+ homeostasis (2Lokuta A.J. Meyers M.B. Sander P.R. Fishman G.I. Valdivia H.H. J. Biol. Chem. 1997; 272: 25333-25338Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 3Meyers M.B. Puri T.S. Chien A.J. Gao T. Hsu P.H. Hosey M.M. Fishman G.I. J. Biol. Chem. 1998; 273: 18930-18935Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar)Sorcin is a penta-EF hand Ca2+-binding protein first identified in cultured fibroblasts selected for resistance to chemother-apeutic agents (4Meyers M.B. Schneider K.A. Spengler B.A. Chang T.D. Biedler J.L. Biochem. Pharmacol. 1987; 36: 2373-2380Crossref PubMed Scopus (49) Google Scholar, 5Van der Bliek A.M. Meyers M.B. Biedler J.L. Hes E. Borst P. EMBO J. 1986; 5: 3201-3208Crossref PubMed Scopus (113) Google Scholar). Subsequent analysis has demonstrated that sorcin is widely expressed in many tissues, including the heart where it is preferentially localized at or near the T-tubules (6Meyers M.B. Pickel V.M. Sheu S.S. Sharma V.K. Scotto K.W. Fishman G.I. J. Biol. Chem. 1995; 270: 26411-26418Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Consistent with this subcellular localization, biochemical studies have demonstrated that sorcin binds directly to both cardiac RyRs and the pore-forming Cav1.2-subunit of the L-type Ca2+ channel (2Lokuta A.J. Meyers M.B. Sander P.R. Fishman G.I. Valdivia H.H. J. Biol. Chem. 1997; 272: 25333-25338Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 3Meyers M.B. Puri T.S. Chien A.J. Gao T. Hsu P.H. Hosey M.M. Fishman G.I. J. Biol. Chem. 1998; 273: 18930-18935Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 6Meyers M.B. Pickel V.M. Sheu S.S. Sharma V.K. Scotto K.W. Fishman G.I. J. Biol. Chem. 1995; 270: 26411-26418Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). The structural basis for the association of sorcin with RyRs remains undetermined; however, it was recently demonstrated that sorcin binds to cardiac L-type Ca2+ channels at a cytoplasmically oriented site on the carboxyl terminus, at or near the calmodulin-binding domain within a region of the Cav1.2 subunit implicated in Ca2+-dependent inactivation (7de Leon M. Wang Y. Jones L. Perez-Reyes E. Wei X. Soong T.W. Snutch T.P. Yue D.T. Science. 1995; 270: 1502-1506Crossref PubMed Scopus (240) Google Scholar, 8Zhou J. Olcese R. Qin N. Noceti F. Birnbaumer L. Stefani E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2301-2305Crossref PubMed Scopus (85) Google Scholar, 9Soldatov N.M. Oz M. O'Brien K.A. Abernethy D.R. Morad M. J. Biol. Chem. 1998; 273: 957-963Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 10Zuhlke R.D. Reuter H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3287-3294Crossref PubMed Scopus (159) Google Scholar, 11Peterson B.Z. DeMaria C.D. Adelman J.P. Yue D.T. Neuron. 1999; 22: 549-558Abstract Full Text Full Text PDF PubMed Scopus (708) Google Scholar, 12Peterson B.Z. Lee J.S. Mulle J.G. Wang Y. de Leon M. Yue D.T. Biophys. J. 2000; 78: 1906-1920Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 13Pitt G.S. Zuhlke R.D. Hudmon A. Schulman H. Reuter H. Tsien R.W. J. Biol. Chem. 2001; 276: 30794-30802Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). Studies of RyRs incorporated into lipid bilayers have revealed that sorcin, applied to the cytoplasmic region of RyRs, inhibits the open probability of the Ca2+ release channel, an effect that is abrogated when sorcin is phosphorylated by protein kinase A (2Lokuta A.J. Meyers M.B. Sander P.R. Fishman G.I. Valdivia H.H. J. Biol. Chem. 1997; 272: 25333-25338Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar).Sorcin has also been implicated in Ca2+ regulation in tissues other than heart, including skeletal muscle (3Meyers M.B. Puri T.S. Chien A.J. Gao T. Hsu P.H. Hosey M.M. Fishman G.I. J. Biol. Chem. 1998; 273: 18930-18935Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) and brain, where sorcin co-localizes with N-methyl-d-aspartate receptors (14Pickel V.M. Clarke C.L. Meyers M.B. J. Comp. Neurol. 1997; 386: 625-634Crossref PubMed Scopus (34) Google Scholar, 15Gracy K.N. Clarke C.L. Meyers M.B. Pickel V.M. Neuroscience. 1999; 90: 107-117Crossref PubMed Scopus (27) Google Scholar) and binds to presenilin 2 (16Pack-Chung E. Meyers M.B. Pettingell W.P. Moir R.D. Brownawell A.M. Cheng I. Tanzi R.E. Kim T.W. J. Biol. Chem. 2000; 275: 14440-14445Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The protein has also been shown to bind to annexin VII and to inhibit annexin VII-mediated chromaffin granule aggregation (17Brownawell A.M. Creutz C.E. J. Biol. Chem. 1997; 272: 22182-22190Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Interestingly, targeted disruption of annexin VII in mice has recently been shown to result in alterations in frequency-induced shortening of isolated cardiac myocytes (18Herr C. Smyth N. Ullrich S. Yun F. Sasse P. Hescheler J. Fleischmann B. Lasek K. Brixius K. Schwinger R.H. Fassler R. Schroder R. Noegel A.A. Mol. Cell. Biol. 2001; 21: 4119-4128Crossref PubMed Scopus (72) Google Scholar).Given the accumulating evidence supporting an important role for sorcin in the regulation of intracellular Ca2+ cycling, we undertook the present study to further explore the expression and function of sorcin in the mammalian heart. Transgenic (TG) mice with cardiac-specific overexpression of sorcin displayed significant reductions in cardiac contractility and relaxation properties, without evidence of hypertrophy or failure; these results were confirmed by studies of single-cell mechanics. Global Ca2+ transients were significantly depressed in sorcin-overexpressing myocytes, consistent with the reduction in contractile function. The amplitude of L-type Ca2+ currents in sorcin-overexpressing myocytes was not different from control cells; however, the time course of inactivation of L-type Ca2+ currents was accelerated, and this effect was confirmed in a Xenopus oocyte expression assay. Finally, using confocal immunomicroscopy, we found that the localization of both sorcin and RyRs was markedly perturbed in failing hearts from spontaneous hypertensive heart failure (SHHF) rats, a model of heart failure in which excitation-contraction coupling is abnormal (19Gomez 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). Together, these data suggest that sorcin modulates cardiac excitation-contraction coupling.EXPERIMENTAL PROCEDURESConstruction of the α-Myosin Heavy Chain-Sorcin Transgene—Human sorcin cDNA was obtained from a cDNA library by gene trapping (Invitrogen) and cloned into the MluI/NotI sites of the pCIneo mammalian expression vector (Promega, Madison, WI). The sorcin sequence was removed with XhoI and SacI and blunt end-ligated into the SalI-HindIII restriction sites of the mouse α-myosin heavy chain promoter expression cassette kindly provided by J. Robbins (The Children's Hospital and Research Foundation, Cincinnati, OH) (20Gulick J. Subramaniam A. Neumann J. Robbins J. J. Biol. Chem. 1991; 266: 9180-9185Abstract Full Text PDF PubMed Google Scholar, 21Rindt H. Subramaniam A. Robbins J. Transgenic Res. 1995; 4: 397-405Crossref PubMed Scopus (36) Google Scholar).Generation of Sorcin-overexpressing TG Mice—A NotI fragment comprised of the α-myosin heavy chain promoter, the protein coding region for human sorcin, and the human growth hormone polyadenylation signal was separated from vector backbone by agarose gel electrophoresis. The fragment was purified with the use of QIAquick (Qiagen) and resuspended for microinjection as previously described (21Rindt H. Subramaniam A. Robbins J. Transgenic Res. 1995; 4: 397-405Crossref PubMed Scopus (36) Google Scholar). Transgenic mice were generated by pronuclear injection at the Mount Sinai School of Medicine Transgenic Mouse Facility according to standard techniques in C57Bl/6 × C3H (B6C3) F1 hybrids. Founder mice were identified by Southern blotting. Transgenic lines were established by breeding founders with nontransgenic littermates, and offspring were genotyped by PCR assay of tail digests using the following primer pair: 5′-GGAAAGTCAGGACTTCACATA-3′ and 5′-AACCCATTGTGCCAGACATATCTC-3′. All animal experiments were approved by the Animal Care Review Boards of the appropriate institutions. All experiments were carried out using mice that were 4–6 months of age.Immunoblotting—Levels of proteins in homogenates of hearts and other tissues were determined by immunoblot analysis, as previously described (3Meyers M.B. Puri T.S. Chien A.J. Gao T. Hsu P.H. Hosey M.M. Fishman G.I. J. Biol. Chem. 1998; 273: 18930-18935Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 6Meyers M.B. Pickel V.M. Sheu S.S. Sharma V.K. Scotto K.W. Fishman G.I. J. Biol. Chem. 1995; 270: 26411-26418Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Briefly, the tissues were lysed by homogenization in a buffer containing 1% Triton X-100, aliquots of soluble material containing 60–100 μg of protein were subjected to electrophoresis, and proteins were transferred to nitrocellulose. After blocking in 5% milk, exposure to primary antibodies for 1 h at room temperature or overnight at 4 °C, and exposure to appropriate horseradish peroxidase-conjugated secondary antibodies, the proteins were detected by enhanced chemiluminescence (Pierce). To determine the abundance of individual calcium regulatory proteins, the blots were scanned and quantified by densitometry, and the signals were normalized by comparison to sarcomeric actin. The data are presented as fold change compared with WT levels. Antibodies raised in rabbits against a COOH-terminal sorcin peptide have been described (6Meyers M.B. Pickel V.M. Sheu S.S. Sharma V.K. Scotto K.W. Fishman G.I. J. Biol. Chem. 1995; 270: 26411-26418Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), and NH2-terminal sorcin peptide antibodies were obtained from Zymed Laboratories (San Francisco, CA). Other antibodies were purchased from Sigma, Affinity Bioreagents (Golden, CO), or Zymed Laboratories Inc.. In a group of experiments, recombinant sorcin (22Meyers M.B. Zamparelli C. Verzili D. Dicker A.P. Blanck T.J. Chiancone E. FEBS Lett. 1995; 357: 230-234Crossref PubMed Scopus (60) Google Scholar) was subjected to enzymatic digestion with endoproteinase Asp-N (Sigma) as previously published (23Zamparelli C. Ilari A. Verzili D. Giangiacomo L. Colotti G. Pascarella S. Chiancone E. Biochemistry. 2000; 39: 658-666Crossref PubMed Scopus (32) Google Scholar).Whole Animal Hemodynamics—A closed chest model was used for hemodynamic assessment. The animals were anesthetized using avertin (2,2,2-tribromoethanol) at 20 μg/g through intraperitoneal injection. Tracheostomy was performed, and the mice were ventilated using a Harvard apparatus ventilator. A 1.4F Millar catheter was introduced via the right carotid artery into the left ventricular cavity. The catheter was interfaced with Ponemah physiology platform software (Gould Instruments, Valley View, OH).Cardiac Myocyte Isolation—The methods used in this laboratory for isolation of adult mouse cardiac myocytes have been published (24Wolska B.M. Solaro R.J. Am. J. Physiol. 1996; 271: H1250-H1255Crossref PubMed Google Scholar). Briefly, the hearts from anesthetized mice were mounted in a Langendorff perfusion system heated to 37 °C and perfused with collagenase (Type 2 collagenase from Worthington Biochemical Corp., Lakewood, NJ) solution saturated with 100% O2 at 37 °C. Isolated myocytes were used within 24 h.Measurement of Fractional Shortening—Isolated myocytes were superfused at (2–3 ml/min at 33 °C) with Tyrode's solution (137 mm NaCl, 5.4 mm KCl, 10 mm HEPES, 1.8 mm CaCl2, 1 mm MgCl2, 0.33 mm NaH2PO4; pH was adjusted to 7.2 with NaOH) on the stage of an inverted microscope (Diaphot 300, Nikon, Tokyo, Japan). The cells were field-stimulated (Grass S88, Grass Telefactor, West Warwick, RI), and edge detection was performed (Crescent Electronics, Sandy, UT) as previously described (25Balaguru 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).Fluorescence Measurements—The cells were placed on the stage of an inverted confocal laser scanning microscope (LSM-510; Carl Zeiss, Inc., Germany), superfused with Tyrode's solution (at 22–24 °C), and field-stimulated (0.5 or 2 Hz). Fluo-3 loading was achieved by a 10-min incubation of the myocytes in Tyrode's solution containing 10 μm of the acetoxymethyl ester form fluo-3 AM (Molecular Probes, Eugene, OR) dissolved in Me2SO, followed by a 15-min wash in Tyrode's solution. The dye was excited by the 488-nm line of an argon laser. The images were acquired in line scan mode (synchronized with the electrical stimulation), with the scan line usually oriented along the long axis of the myocyte, avoiding the cell nuclei. Optical settings were similar as described before (26Zhou Y.Y. Song L.S. Lakatta E.G. Xiao R.P. Cheng H. J. Physiol. 1999; 521: 351-361Crossref PubMed Scopus (57) Google Scholar). Image processing, data analysis, and presentation were performed using Scion Image (Scion Corp., Frederick, MD), Excel (Microsoft), and OriginPro (OriginLab Corp., Northampton, MA). Briefly, the background fluorescence was subtracted from each image and fluorescence (F) at diastole was defined as F 0. The profiles were constructed along the time axis of the line scan, and the relative increase in fluorescence (F/F 0) was plotted as a function of time.Measurement of Whole Cell Ca2 + Currents—The methods for measurement of currents in adult mouse cardiac myocytes have been published (27Heubach J.F. Graf E.M. Molenaar P. Jager A. Schroder F. Herzig S. Harding S.E. Ravens U. Br. J. Pharmacol. 2001; 133: 73-82Crossref PubMed Scopus (19) Google Scholar). Briefly, whole cell voltage-clamp technique was applied using an EPC9/2 amplifier to record membrane currents at room temperature (23 °C); compensation was adjusted automatically. Command pulse timing and amplitude was controlled using Pulse + PulseFit software (version 8.53; HEKA), whereas acquiring current data and Origin 6.1 was used for data analysis. Patch pipettes (tip resistance, 1.5–5.5 MΩ) were fabricated on a micropipette puller (model P-97; Sutter Instrument Company, Novato, CA) and fire-polished. The composition of the bath solution was 137 mm NaCl, 5.4 mm CsCl, 2.0 mm CaCl2, 1.25 mm MgCl2, 10.0 mm HEPES, 10.0 mm glucose (the pH was adjusted to 7.4 with NaOH). The pipette solution had the following composition 140 mm CsCl, 4 mm MgCl2, 10 mm HEPES, 4 mm Na2ATP, and either 0.5 or 10 mm EGTA (the pH was adjusted to 7.3 with CsOH). The myocytes were clamped at a holding potential of –80 mV, prepulsed for 50 ms to –40 mV to inactivate Na+ currents, and subjected to 500-ms depolarizing pulses to potentials between –40 and +80 mV in 10-mV increments. Peak currents at +10 mV are reported. The peak current amplitude was determined as the difference between the peak inward current and the current at the end of the depolarizing pulse. To account for variability in cell size, the membrane current was expressed as current density (pA/pF). The inactivation time course was fit to a two exponential function from which fast and slow time constants (τ) were derived. In addition, the time to 50% decay of peak current (t 50 inact) was determined.Measurement of Current in Oocytes—Oocytes were isolated from Xenopus laevis using collagenase as described previously (28Chan K.W. Sui J.L. Vivaudou M. Logothetis D.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14193-14198Crossref PubMed Scopus (96) Google Scholar). Calcium channel subunit cDNA constructs in the pAGA2 oocyte expression vector (29Sanford J. Codina J. Birnbaumer L. J. Biol. Chem. 1991; 266: 9570-9579Abstract Full Text PDF PubMed Google Scholar) and sorcin cDNA constructs in a pCR3 vector (Invitrogen) were linearized and subjected to in vitro transcription using the mMessage mMachine kit (Ambion, Austin, TX). The resulting cRNAs were quantified by comparison of two dilutions to a standard on a formaldehyde gel. For recordings from L-type channels, cRNA of α1c, α2δ, and β3 subunits of the Ca2+ channels were injected into Xenopus oocytes at a ratio of 3:1:4, respectively, with a total of 4 ng of cRNA/oocyte. Following injection, the oocytes were kept in an 18 °C incubator, and two electrode voltage clamp recordings were performed 2 days after cRNA injections. The recording solutions contained in 50 mm sodium acetate, 40 mm barium acetate, 2 mm potassium acetate, 5 mm HEPES (pH was adjusted to 7.5 with Ba(OH)2). The voltage protocol for the recording of Ba2+ currents consisted of voltage steps of 300-ms duration from a holding potential of –100 mV to +60 mV in 10-mV steps, repeated at a frequency of 0.5 Hz. The rate of inactivation was quantified as the fraction of Ba2+ current remaining at the end of the 300-ms pulse compared with the peak current.Immunofluorescent Microscopy—Tissues from C57BL/6 WT and TG mice and from normal 17–18-month-old Sprague-Dawley and Wistar-Furth rats and 17–18-month-old SHHF rats in overt heart failure were studied. Each SHHF animal exhibited the typical signs of heart failure including cardiac hypertrophy, pulmonary congestion, and pleural effusions, ascites, and hepatic congestion (19Gomez 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). For analysis of tissues, the animals were sacrificed after inhalation of metofane according to approved protocols. The hearts were quickly excised, washed in cold PBS, and stored in 30% sucrose overnight. Tissue blocks were placed in Tek O.C.T. compound (Electron Microscopy Sciences, Torrance, CA) and snap frozen in liquid nitrogen chilled iso-pentane for 10 s, and 5-micron sections were prepared for immunolabeling. The sections were fixed in acetone at –20 °C for 10 min, washed in PBS, and blocked in 1% bovine serum albumin in PBS for1hat room temperature before incubation in primary and secondary antibodies. All of the antibodies were diluted in 1% bovine serum albumin, and PBS was used for washes after each antibody exposure. Rabbit polyclonal sorcin antibody (6Meyers M.B. Pickel V.M. Sheu S.S. Sharma V.K. Scotto K.W. Fishman G.I. J. Biol. Chem. 1995; 270: 26411-26418Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) was used at 37 °C for 1 h followed by Cy3-conjugated anti-rabbit secondary antibody (Zymed Laboratories) at room temperature for 1 h. Mouse monoclonal RyR antibody (Sigma) was used at 4 °C for 16 h followed by fluorescein isothiocyanate-conjugated anti-mouse secondary (Sigma) for 1 h at room temperature. Confocal images of rat tissues were obtained with a Leica TCS-SP Spectral Confocal microscope in an inverted configuration. Illumination for fluorescein isothiocyanate was obtained by argon laser at 488 nm with emissions collected at 505–550 nm and for Cy3 by krypton laser at 568 nm with emissions collected at 610–700 nm. The objective was 40×1Na oil PL FL 1–0.5 with zoom factors of 2.45 (x and y voxel sizes 0.2 × 0.2 μm). Images of immunostained mouse cardiac myocytes were obtained by fluorescent light microscopy (Diaphot 300, Nikon) with a 40× objective.Statistics—The data are expressed as the mean values ± S.E., and p values <0.05 were considered significant.RESULTSCharacterization of Sorcin-overexpressing TG Mice—To explore the function of sorcin in vivo, we generated transgenic mice in which sorcin was overexpressed in the heart using the well characterized α-myosin heavy chain promoter. Four independent founders were identified by Southern blotting. Transgene expression, as determined by Northern blot analysis using total cardiac RNA, was examined in three of these lines and found to be most abundant in line 24, moderate in line 23, and weakest in line 6, as shown in Fig. 1A. Accordingly, we selected the line with intermediate levels of expression (line 23), for further characterization. Sorcin protein expression, as determined by Western blotting, was increased 20-fold in TG hearts from line 23 compared with hearts from WT littermates (Fig. 1B). As previously reported (3Meyers M.B. Puri T.S. Chien A.J. Gao T. Hsu P.H. Hosey M.M. Fishman G.I. J. Biol. Chem. 1998; 273: 18930-18935Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), sorcin exists in the rodent heart as both the predicted 22-kDa full-length form and a more abundant ∼18-kDa truncated species (hereafter referred to as t-sorcin), which appears to result from proteolytic cleavage within the NH2 terminus near aspartate residue 33 (24Wolska B.M. Solaro R.J. Am. J. Physiol. 1996; 271: H1250-H1255Crossref PubMed Google Scholar). Using anti-peptide antibodies specific for epitopes from the amino or carboxyl terminus of sorcin, we found that only the full-length, 22-kDa form of sorcin was overexpressed in transgenic hearts, whereas the abundance of the 18-kDa t-sorcin form was essentially equivalent in TG and WT hearts (Fig. 1B).To test whether overexpression of sorcin influenced the abundance of other cardiac Ca2+-regulatory proteins, we compared expression levels in a series of TG and WT control hearts (n = 8 from each group). There was no significant change in expression of Cav1.2, the pore-forming subunit of the L-type Ca2+ channel (0.95 ± 0.05), RyR (1.05 ± 0.05), SERCA2A (0.70 ± 0.34), phospholamban (1.40 ± 0.30), or calsequestrin (1.33 ± 0.46) in TG compared with WT hearts (p is not significant for all comparisons). Representative blots are shown in Fig. 1C.We next examined the localization of overexpressed sorcin in dissociated adult myocytes from transgenic hearts, using immunocytochemical means and a limiting antibody dilution technique (Fig. 1D). At a primary C terminus-specific antibody dilution of 1:5000, sorcin was observed at the Z-lines in both WT and TG myocytes. At a dilution of 1: 25,000, sorcin was no longer detectable in WT myocytes but was still easily detectable in TG cells, consistent with its marked overexpression. Thus, not only was sorcin overexpressed in the TG hearts, it appeared to target appropriately.Phenotypically, transgenic mice developed normally and showed no evidence of morbidity and no early mortality. Grossly, there was no cardiac hypertrophy, and this finding was confirmed histologically (not shown) and by measuring heart weight/body weight ratios, which were no different between WT and TG (5.5 ± 0.5 mg/g versus 5.6 ± 0.6 mg/g, respectively; n = 5).In Vivo Physiology—We next examined whether constitutive overexpression of sorcin in the heart influenced cardiac contractile function. Heart rate (399 ± 32 beats/min versus 481 ± 94 beats/min) and peak left ventricular systolic (113 ± 11 mm Hg versus 129 ± 15 mm Hg) were modestly reduced in TG mice compared with WT controls. Left ventricular end diastolic pressure (7.5 ± 5.8 mm Hg versus 6.9 ± 6.5 mm Hg) was not statistically different in TG and WT mice. Interestingly, despite the absence of demonstrable anatomic abnormalities such as hypertrophy or chamber dilatation in TG mice, we observed a highly significant reduction in left ventricular contractile and relaxation parameters compared with WT littermate controls. Both dP/dT max (6620 ± 1416; n = 9 versus 11,101 ± 4193; n = 9; p = 0.013) and dP/dT min (6663 ± 1344 versus 9902 ± 2784; n = 9; p < 0.05) were significantly reduced, and the time constant of relaxation (τ) was significantly prolonged (8.01 ± 1.34 versus 6.13 ± 1.29 ms; n = 9; p < 0.05), in TG animals compared with WT controls. These data are summarized in Table I.Table IIn vivo hemodynamicsWTTGHeart rate (beats/min)481 ± 94399 ± 32ap < 0.05.Peak systolic pressure (mm Hg)129 ± 14.9112 ± 10.9ap < 0.05.LV end diastolic pressure (mm Hg)6.9 ± 6.57.5 ± 5.8dP/dt max11101 ± 41936629 ± 1416ap < 0.05.dP/dt min9902 ± 27836663 ± 1344ap < 0.05.a p < 0.05. Open table in a new tab Fractional Shortening and Ca2 + Transients in Isolated Myocytes—To explore the cellular mechanisms accounting for the depression of cardiac contractility, we measured cell shortening and Ca2+ transients in isolated adult myocytes from TG and WT mice field-stimulated over a range of frequencies (0.5–3 Hz), as shown in Fig. 2. WT myocytes displayed a negative force-frequency relationship typical of adult murine cardiomyocytes. At all stimulation rates, the twitch amplitude was depressed in TG myocytes compared with WT cells, and the force-frequency relationship was flat (Fig. 2A). In addition, the twitch rise and decay times were prolonged (Fig. 2B; e.g. at 2 Hz the rise time was 30.1 ± 2.2 ms versus 22.8 ± 1.6 ms for TG and WT, respectively, and the corresponding decay times were 54.7 ± 5.2 ms versus 37.7 ± 3.2 ms; p < 0.05), suggesting abnormalities in both contraction and relaxation and consistent with the in vivo intact heart analysis (Table II).Fig. 2Single cell mechanics and Ca2+ transients in isolated adult cardiac myocytes. A, summary data of fractional shortening during field stimulation over a range of frequencies in TG and WT myocytes. At each frequency, there is a significant depression of twitch amplitude in the TG cells. B, representative traces of cell length during field stimulation at 0.5 Hz in TG and WT myocytes. C, line scans demonstrating Ca2+ transients in fluo-3 loaded WT (upper panels) and TG (lower panels) cells stimulated at 0.5 Hz (left panels) and 2.0 Hz (right panels). D, Ca2+ transient presented as plots of integrated pixel intensity versus time for WT (black line) and TG (blue line) myocytes.Vie
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