Cardiovascular Research

Cardiovascular Research 2003 59(2):369-379; doi:10.1016/S0008-6363(03)00432-2
© 2003 by European Society of Cardiology

This Article Abstract FREE Full Text (PDF) E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Disclaimer Google Scholar Articles by Kirchhefer, U. Articles by Jones, L. R. Search for Related Content PubMed PubMed Citation Articles by Kirchhefer, U. Articles by Jones, L. R. Social Bookmarking          What's this?

Copyright © 2003, European Society of Cardiology

Impaired relaxation in transgenic mice overexpressing junctin

Uwe Kirchhefer^a,*, Joachim Neumann^a, Donald M. Bers^b, Igor B. Buchwalow^c, Larissa Fabritz^d, Gabriela Hanske^a, Isabel Justus^a, Burkhard Riemann^e, Wilhelm Schmitz^a and Larry R. Jones^f

^aInstitut für Pharmakologie und Toxikologie, Westfälische Wilhelms-Universität, Domagkstraße 12, 48149 Münster, Germany
^bDepartment of Physiology, Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153, USA
^cMedizinische Klinik B und Zentrale Projektgruppe Ultrastrukturforschung am Gerhard-Domagk-Institut für Pathologie, Westfälische Wilhelms-Universität, 48149 Münster, Germany
^dMedizinische Klinik und Poliklinik C, Westfälische Wilhelms-Universität, 48149 Münster, Germany
^eKlinik und Poliklinik für Nuklearmedizin, Westfälische Wilhelms-Universität, 48149 Münster, Germany
^fDepartment of Medicine, Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA

^* Corresponding author. Tel.: +49-251-835-5510; fax: +49-251-835-5501. kirchhef{at}uni-muenster.de

Received 17 December 2002; accepted 15 April 2003

	Abstract

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
Objective: Junctin is a major transmembrane protein in cardiac junctional sarcoplasmic reticulum, which forms a quaternary complex with the ryanodine receptor (Ca²⁺ release channel), triadin, and calsequestrin. Methods: To better understand the role of junctin in excitation–contraction coupling in the heart, we generated transgenic mice with targeted overexpression of junctin to mouse heart, using the -MHC promoter to drive protein expression. Results: The protein was overexpressed 10-fold in mouse ventricles and overexpression was accompanied by cardiac hypertrophy (19%). The levels of two other junctional SR-proteins, the ryanodine receptor and triadin, were reduced by 32% and 23%, respectively. However, [³H]ryanodine binding and the expression levels of calsequestrin, phospholamban and SERCA2a remained unchanged. Cardiomyocytes from junctin-overexpressing mice exhibited impaired relaxation: Ca²⁺ transients decayed at a slower rate and cell relengthening was prolonged. Isolated electrically stimulated papillary muscles from junctin-overexpressing hearts exhibited prolonged mechanical relaxation, and echocardiographic parameters of relaxation were prolonged in the living transgenic mice. The amplitude of caffeine-induced Ca²⁺ transients was lower in cardiomyocytes from junctin-overexpressing mice. The inactivation kinetics of L-type Ca²⁺ channel were prolonged in junctin-overexpressing cardiomyocytes using Ca²⁺ or Ba²⁺ as charge carriers. Conclusion: Our data provide evidence that cardiac-specific overexpression of junctin is accompanied by impaired myocardial relaxation with prolonged Ca²⁺ transient kinetics on the cardiomyocyte level.

KEYWORDS bp, base pairs; EGTA, ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid; HEPES, (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]); PAGE, polyacrylamide gel electrophoresis; SDS, sodium n-dodecyl sulphate; SR, sarcoplasmic reticulum

	1. Introduction

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
In cardiac muscle, depolarization during an action potential leads to the opening of sarcolemmal voltage-dependent L-type Ca²⁺ channels. The Ca²⁺ influx through these channels triggers the release of Ca²⁺ from the sarcoplasmic reticulum (SR). The release of Ca²⁺ from the SR provides the main amount of Ca²⁺ needed for muscle contraction [1]. Ca²⁺ influx leads to opening of the ryanodine receptor in the junctional SR [2]. The increase in cytosolic Ca²⁺ terminates the opening of the ryanodine receptor [3]. After activation of the myofilaments, the muscle relaxes because Ca²⁺ is actively transported back into the free SR (situated around the myofilaments) by a SR-Ca²⁺ATPase (SERCA2a). The function of Ca²⁺ uptake into the free SR has been very intensely studied with the help of transgenic and gene ablation experiments in mice [4]. On the other hand, the release of Ca²⁺ at the junctional SR is less well understood.

The junctional SR is enriched in the ryanodine receptor, junctin, triadin, and calsequestrin. The ryanodine receptor forms a tetrameric structure, comprising the so-called feet as visualized by electron microscopy [5]. Initial studies in skeletal muscle indicated a regulation of the ryanodine receptor by proteins in the junctional SR. First calsequestrin, a high capacity, low affinity Ca²⁺ binding protein in the residing of the lumen of junctional SR, was identified [6]. Ca²⁺ bound to calsequestrin changes the conformation of calsequestrin and increases the amount of Ca²⁺ released from the SR [7], providing evidence for a functional interplay between calsequestrin and the ryanodine receptor. Electron microscopic studies and biochemical work indicated that calsequestrin does not directly bind to the SR [8,9]. The binding is mediated by anchoring proteins. Using ¹²⁵I-labeled calsequestrin in overlay of cardiac membranes bound to nitrocellulose, only a few major calsequestrin binding proteins are identified [9,10]. The two most prominent calsequestrin binding proteins are triadin and junctin, which were purified and cloned. Junctin migrates as a 26-kDa protein. Canine junctin has 210 amino acids and is highly enriched in the junctional SR in both cardiac and skeletal muscle. The N-terminal amino acids 1–22 are predicted to face the cytoplasm. Junctin like triadin has a single transmembrane domain, comprised of amino acids 23–44 [10]. The residues 45–210, which are responsible for binding of calsequestrin, are located in the SR-lumen [9]. At physiological Ca²⁺ in the SR a considerable fraction of the lumenal calsequestrin is predicted to bind to junctin, but the interaction of junctin and calsequestrin is reduced at increasing Ca²⁺ concentrations. Junctin also binds to triadin, but this interaction is independent of Ca²⁺ concentrations, at least in vitro [9]. Junctin can bind to itself and to the ryanodine receptor [9]. Thus, junctin may act as a scaffold to collect and tether diverse molecules at the junctional SR. It is unknown whether junctin plays an active role in regulating cardiac SR-Ca²⁺ release. However, several studies implicated junctin and its isoforms in mediating skeletal muscle Ca²⁺ release [11–13].

Guo and Campbell noticed an interaction of the lumenal domain of skeletal muscle triadin with the ryanodine receptor and calsequestrin, which occurred in a Ca²⁺-dependent manner [14]. In cardiac muscle, the primary isoform of triadin expressed is triadin 1 [15]. Junctin and triadin are derived from different genes but structurally and biochemically exhibit a number of similarities [16,17]. Several short runs of identical sequences and sequence homologies between junctin and triadin were noted [10]. Junctin may bind more strongly to calsequestrin than triadin and may therefore be of greater physiological relevance for Ca²⁺ sensing of the ryanodine receptor [9]. Beyond simply anchoring calsequestrin to the ryanodine receptor, junctin may affect the channel activity of the ryanodine receptor directly. An active role in SR-Ca²⁺ release in the heart was suggested for triadin 1. First experiments, in which skeletal muscle triadin was applied to purified ryanodine receptor in planar lipid bilayers, showed that the channel activity was inhibited [18,19]. In addition, the forced overexpression of triadin 1 in ventricle was accompanied by altered Ca²⁺ transient kinetics and impaired relaxation [20]. The functional testing of atrium with triadin 1 overexpression revealed that the SR-Ca²⁺ loading was increased [21].

In the present study, we characterize transgenic mice with cardiac-specific overexpression of junctin [22]. Our experiments show that this overexpression results in impaired relaxation with altered Ca²⁺ transient kinetics. The decreased protein expression of triadin and the ryanodine receptor and other factors might be involved in the observed contractile alterations.

	2. Methods

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
2.1 Experimental animals
Transgenic mice overexpressing canine junctin were generated as previously described [22]. Junctin-overexpressing mice were identified by polymerase chain reaction [23]. All experiments were carried out on mice 16–20 weeks of age. Animals were handled and maintained according to approved protocols of the animal welfare committees of the University of Münster, Germany, and Indiana University, USA.

2.2 Immunohistochemical staining
Paraffin sections of formaldehyde-fixed hearts from wild-type and junctin-overexpressing mice were mounted on polysine microslides (Menzel Gläser, Braunschweig, Germany). For the antigen retrieval, dewaxed and rehydrated tissue sections were immersed in 10 mM citric acid, pH 6.0, and boiled in a pressure cooker at an operating pressure of about 103 kPa/15 psi for 2 min. After cooling, the slides were removed, quickly washed in distilled water and transferred to PBS solution. PBS was used for all following dilutions and washing steps. Then, sections were encircled with a water-repellent PAP-pen (Dianova, Hamburg, Germany) and rinsed with PBS. After blocking non-specific binding sites (Fc-receptors) with BSA-c basic blocking solution (1:10, Aurion, Wageningen, Netherlands), sections were immunolabeled overnight at 4°C with monoclonal mouse primary antibody 5D8 recognizing canine junctin [22]. Immunolabeling with this antibody, which was raised in mouse, is possible only after its haptenylation. This was carried out employing N-hydroxy-succinimide esters of biotin (Molecular Probes) following the guidelines recommended by the manufacturers. Biotinylated primary antibody was visualized with FITC-streptavidin (1:300). Finally, samples were counterstained for 15 s with DAPI (5 µg/ml PBS; Sigma) and mounted with Vectashield (Vector Laboratories, USA). Immunostained sections were examined on a Zeiss Axiophot 2 microscope equipped with appropriate filters. Separate images for DAPI staining, FITC-immunolabeling and for autofluorescence (e.g. caused by myocardium or erythrocytes) were captured digitally into color-separated components using an AxioCam digital microscope camera and AxioVision multi channel image processing (Carl Zeiss Vision, Jena, Germany). The blue (for DAPI), red (for autofluorescence), and green (for FITC) components were merged, and composite images were imported as JPG files into PhotoImpact 3.0 (Ulead Systems Inc., Torrance, CA, USA) for analysis on Power PC.

2.3 Northern blotting
Northern blots containing 20 µg total RNA isolated from ventricular tissue were hybridized with ³²P-radiolabeled cDNA probes following standard protocols [24]. The cDNA probes encoded for canine junctin and mouse atrial natriuretic factor (ANF). For generation of the canine junctin cDNA probe, forward and reverse primers were chosen from the coding region [10]. The ANF cDNA probe was obtained as recently described [20].

2.4 Biochemical analyses
Isolated hearts from wild-type or junctin-overexpressing mice were homogenized at 4°C for 90 s in 1 ml of solution containing 10 mM histidine (pH 7.4) and 0.25 M sucrose, using a Polytron PT-10 (Kinematica, Lucerne, Switzerland). Protein concentrations were determined according to the method of Lowry et al. [25]. Homogenate protein was solubilized at room temperature in 5% SDS-buffer containing 62.5 mM Tris–HCl (pH 6.8), 5% glycerol, 40 mM dithiothreitol, and a trace of bromphenol blue. Then 50 µg or 200 µg (for detection of ryanodine receptor) of homogenate protein was separated on 8% or 5% SDS–PAGE, respectively [26]. Thereafter, quantitative immunoblotting was used to determine the expression level of Ca²⁺ handling proteins of the free SR, like SERCA2a [10], calsequestrin [27], and phospholamban [28], and of the junctional SR, like triadin 1 [15], and the ryanodine receptor [9].

2.5 [³H]Ryanodine binding
Preparation of crude homogenates from individual hearts of wild-type and junctin-overexpressing mice and detection of [³H]ryanodine binding to the ryanodine receptors were carried out as reported [20].

2.6 β-Adrenergic receptor density
Microsomes were prepared by homogenizing individual ventricles from junctin-overexpressing and wild-type hearts at 4°C for 90 s in 1 ml of a buffer A containing 10 mM EDTA, 10 mM HEPES, 0.1 mM benzamidine (pH 7.4), using a Polytron PT 3000 (Kinematica, Lucerne, Switzerland). Homogenates were centrifuged at 45 000xg_max for 15 min at 4°C. The pellets were resuspended in 10 ml of a buffer B containing 1 mM EDTA, 10 mM HEPES, 0.1 mM benzamidine (pH 7.4) and recentrifuged at 45 000xg_max for 15 min at 4°C. The pellets were resuspended in 1 ml of buffer B and centrifuged at 10 000xg_max for 10 min at 4°C. The supernatants were recentrifuged at 45 000xg_max for 15 min at 4°C. The pellets, partially enriched in membranes, were resuspended in 50 mM Tris–HCl, 5 mM MgCl₂, pH 7.4, and stored frozen at –80°C. Membranes were used for the measurement of β-adrenergic receptor (AR) density. Total β-AR density was determined by incubating 15 µg of membrane protein with increasing concentrations of [¹²⁵I]ICYP (1–300 pM) in a buffer of 10 mM Tris, 154 mM NaCl, 0.1 mM ascorbic acid (pH 7.4). The nonspecific binding was measured in the presence of 20 µM alprenolol. Reactions were conducted at 37°C for 60 min. Samples were filtered onto Whatman GF/B filters, washed, and the membrane-bound activity was determined in a -scintillation counter. The maximum number of binding sites (B_max) was calculated from plots according to the method of Scatchard [29].

2.7 Measurement of Ca²⁺ transient [Ca]_i
Cardiomyocytes were isolated from wild-type and junctin-overexpressing mouse hearts by a standard enzymatic digestion procedure [30]. Intracellular Ca²⁺ transients of Indo-1/AM loaded cardiomyocytes were determined and analyzed as described [20]. Briefly, Indo-1 signal was recorded at room temperature from electrically stimulated (0.5 Hz) single cardiomyocytes using a dual-emission microfluorescence system (Photon Technologies Inc., South Brunswick, NJ, USA). Cardiomyocytes were excited at 365 nm and the emitted fluorescence was recorded at 405 and 495 nm. Data were acquired from steady-state Ca²⁺ transients at 2.5 mM [Ca²⁺]₀ for each cardiomyocyte and analyzed with FELIX 1.1 software (Photon Technologies Inc., South Brunswick, NJ, USA). To assess the SR-Ca²⁺ load, caffeine was used as an established tool. First, cardiomyocytes were stimulated at 0.5 Hz, superfused with a Tyrode solution [20]. After a 15-s rest period to achieve maximal SR-Ca²⁺ load, caffeine-induced Ca²⁺ release was initiated by rapid application of 10 mM caffeine (in Tyrode solution) within 4 ms using a fast solution switching system (Warner Instrument Corp., Hamden, CT, USA). Caffeine application was maintained for 60 s. This long exposure of caffeine prevented the SR from resequestering released Ca²⁺. The peak amplitude, the decay, and the area under the curve of the caffeine-induced Ca²⁺ transients [Ca]_i, were determined.

2.8 Cardiomyocyte contractile parameters
The video edge detection of wild-type and junctin-overexpressing cardiomyocytes was conducted simultaneously with [Ca]_i measurements at room temperature as described [20]. Cardiomyocytes were electrically stimulated at 0.5 Hz. The extracellular Ca²⁺ concentration was adjusted to 2.5 mM. The steady-state twitches of cardiomyocytes were recorded and analyzed.

2.9 Whole-cell L-type Ca²⁺ current (I_Ca)
L-type Ca²⁺ channel currents were measured in isolated cardiomyocytes using the whole-cell configuration of the patch-clamp technique [20]. All experiments were carried out at room temperature and under conditions that suppress Na⁺ and K⁺ currents. The extracellular buffer contained 2 mM CaCl₂ and the micropipette was filled with buffer including 1 mM EGTA. Where indicated, 2 mM BaCl₂ instead of Ca²⁺ was used in the extracellular solution (I_Ba). I_Ca was recorded at different potentials, starting from a holding potential of –40 mV. The inactivation kinetics of I_Ca were studied at a test potential of +10 mV. Cell capacitance and L-type Ca²⁺ channel current were recorded continuously with an L/M-PC amplifier (LIST-Electronic, Darmstadt, Germany). Data acquisition and analyses were realized with ISO2 software (MFK, Niedernhausen, Germany).

2.10 Measurement of contractility of papillary muscles
Right papillary muscles were dissected from isolated hearts of wild-type and junctin-overexpressing mice and mounted in a tissue bath (0.1 ml). The diameter of the papillary muscles was <0.5 mm to prevent anoxic cores. The origin of the papillary muscles was fixed with a fine suture to a hook in the tissue bath, whereas the tendon part was attached to a force transducer (AE801, SensoNor, Horten, Norway) held by a micromanipulator (MN-3333, Leica, Bensheim, Germany). Papillary muscles were perfused at a flow rate of 10 ml/min with a Tyrode solution composed of (in mM) 140 NaCl, 4 KCl, 1 MgCl₂, 2 CaCl₂, 5 HEPES, and 10 glucose (pH 7.4), continuously gassed with 100% O₂. The Tyrode solution contained 30 mM 2,3-butanedione monoxime during the dissection of the papillary muscle from the ventricle. Papillary muscles were stimulated at 0.5 Hz (Grass stimulator, Quincy, MA, USA) and maintained at room temperature. Signals were amplified (FMI, Ober-Beerbach, Germany), transformed (PowerLab 2/20, ADInstruments, Australia), and stored on a computer. Isoproterenol was applied for 5 min (1 µM). Force of contraction and the first derivative of developed tension (+dF/dt and –dF/dt) were measured and monitored continuously. Time to peak tension was calculated as time from 10% of peak contraction to peak contraction. Time of relaxation was determined as time from peak tension to 90% relaxation.

2.11 Echocardiography and Doppler studies
Left ventricular M-mode and Doppler flow measurements were carried out on wild-type and junctin-overexpressing mice. Mice were sedated with ketamine (25 mg/kg) and xylazine (10 mg/kg). This protocol allows spontaneous breathing. Care was taken to keep body temperature of the mice constant. The anesthetic agent was prewarmed and after intraperitoneal injection, the animals were placed under a warming lamp. Warm depilation cream and warm ultrasonic gel were used. During echocardiography, animals were placed on a warming plate. Temperature as documented at the end of the experiment was above 36°C. Bradycardia can be caused by the xylazine component of the anesthesia. For assessment of diastolic function, heart rates below 450 bpm allow discrimination of the early and late phase of filling of the left ventricle by Doppler measurements. At higher rates, there is usually fusion of waves, impairing measurements of deceleration time. In addition, we noted a tendency to lower heart rates in junctin-overexpressing mice. Thus, care was taken to compare deceleration time of different genotypes at comparable heart rates. Therefore, wild-type mice with higher heart rates had to be excluded from analysis, rendering overall heart rates even lower. Measurements were conducted with a commercially available echocardiographic system (Hewlett-Packard Sonos 5500) equipped with a 15 MHz linear transducer for two-dimensional and M-mode imaging and a 12 MHz transducer for Doppler measurements. The parasternal long and short axes were obtained. The fractional shortening of the heart was calculated from the M-mode left ventricular (LV) diameters as (LVEDD–LVESD)/LVEDDx100, where LVEDD was the end-diastolic diameter and LVESD the end-systolic diameter. In addition, Doppler flow measurements of aortic outflow and mitral inflow were carried out.

2.12 Materials
[³H]Ryanodine, [-³²P]dCTP, and ¹²⁵I-labeled protein A were obtained from DuPont NEN (Boston, MA, USA). Indo-1/AM was supplied by Sigma. All other chemicals were of reagent-grade.

2.13 Statistical analysis
Data are reported as means±S.E.M. Statistical differences between the different types of mice were calculated by ANOVA followed by Bonferroni's t-test. P<0.05 was considered significant.

	3. Results

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
3.1 Characterization of junctin-overexpressing mice
The overexpression of canine junctin in myocardium was achieved using the -myosin heavy chain promoter [31]. The transgenic mice exhibited a 10-fold cardiac-specific overexpression of junctin [22]. This is consistent with the high expression of canine junctin mRNA observed in ventricular tissue of junctin-overexpressing mice (Fig. 1A). Here, we used a ³²P-labeled cDNA probe, which was specific for dog junctin. Interestingly, the overexpression of junctin was associated with mild hypertrophy as indicated by a higher heart weight (Table 1). The body weight was unchanged between both groups. Thus, the heart weight/body weight ratio (i.e. the relative heart weight) was increased by 19% in junctin-overexpressing mice. The hypertrophy observed was accompanied by a twofold increase in ANF mRNA expression in left ventricle of junctin-overexpressing mice (Fig. 1B). However, fibrosis was not detected in junctin-overexpressing mice. The life expectancy was comparable between junctin-overexpressing and wild-type mice in contrast to calsequestrin-overexpressing mice [32]. Immunohistochemical staining of formaldehyde-fixed ventricular sections using the mouse monoclonal antibody 5D8 revealed abundant expression of the transgene in junctin-overexpressing cardiomyocytes (Fig. 2). A combination of an intracellular longitudinal and transverse linear pattern of canine junctin expression in the cardiomyocytes was consistent with that which has been described for antigens localized to the free and junctional SR as reported by Hu et al. [33] and by Buchwalow and co-workers [34].

View larger version (30K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Overexpression of junctin and ANF mRNA expression. Shown are the expression of canine junctin (A) and atrial natriuretic factor (B) mRNA in transgenic mouse hearts. Total RNA (20 µg) prepared from wild-type (WT) and junctin-overexpressing (JCN) mouse hearts was separated electrophoretically, transferred to a nylon membrane, and then hybridized with canine junctin and mouse atrial natriuretic factor (ANF) specific radioactive cDNA probes.

 

View this table: [in this window] [in a new window]   Table 1 Morphometric parameters of junctin-overexpressing mice

 

View larger version (66K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Immunohistochemical detection of transgenic junctin. Paraffin sections of formaldehyde-fixed left ventricles from wild-type (WT) and junctin-overexpressing mice (JCN) were immunoreacted with monoclonal mouse primary antibody 5D8 recognizing canine junctin. The biotinylated primary antibody was visualized with FITC-streptavidin. Finally, nuclei were counterstained with DAPI. Immunostained sections were examined on a microscope equipped with appropriate filters. Separate digital images for blue DAPI staining (nuclei), green FITC-immunolabeling (canine junctin), and for red autofluorescence (myocardium and erythrocytes) were obtained separately and later merged as described in Methods.

 
3.2 Expression of Ca²⁺ regulatory SR-proteins and β-AR signaling
The expression of Ca²⁺ regulatory proteins located at the junctional or free SR was tested by immunoblotting. The endogenous mouse junctin (23-KDa protein) was down-regulated by 65% in the transgenic mouse hearts overexpressing canine junctin (26-KDa protein), but still allowing a 10-fold overexpression of the total junctin. The expression level of triadin, a protein which shares identical regions in its amino acid sequence with junctin, was reduced by 23% (Table 2). Remarkably, junctin was the protein most reduced in ventricular homogenates of triadin 1-overexpressing mice [20]. Thus, our data provide further evidence that the expression of both proteins is functionally coupled. In addition, the protein expression of the ryanodine receptor was down-regulated by 32% in junctin-overexpressing hearts. However, [³H]ryanodine binding in homogenates was not significantly different between junctin-overexpressing (B_max=423±46 fmol/mg) and wild-type hearts (B_max=446±56 fmol/mg; n = 6, P>0.05). Because ryanodine binds only to open ryanodine receptors [35], the unaltered [³H]ryanodine binding in homogenates of junctin-overexpressing hearts might also reflect fewer ryanodine receptors with slightly higher open probability. The protein expression of calsequestrin, the Ca²⁺ storage protein in the lumen of the junctional SR, was identical in both groups. Furthermore, the expression levels of SERCA2a and phospholamban, proteins located at the free SR, remained unchanged between junctin-overexpressing and wild-type hearts (Table 2). Cardiac hypertrophy is characterized by a reduced expression of total β-ARs. Here, we determined the β-AR density using [¹²⁵]ICYP binding in membrane preparations. The total density of β-ARs was comparable in preparations from junctin-overexpressing and wild-type hearts (45.3±4.1 fmol/mg in transgenic vs. 47.2±3.1 fmol/mg in wild-type, n = 6).

View this table: [in this window] [in a new window]   Table 2 Levels of SR-proteins in homogenates from wild-type and junctin-overexpressing mouse hearts

 
3.3 In vivo measurement of cardiac performance
The in vivo ventricular function of junctin-overexpressing mice was measured by transthoracic echocardiography. Parameters were measured by M-mode and Doppler echocardiography (Table 3). The intraventricular septum thickness was unaltered in junctin-overexpressing mice. In addition, the left ventricular end-systolic and end-diastolic diameters (like the fractional shortening) were not different between both groups. Mean left ventricular end-diastolic diameter was increased by 5% in junctin-overexpressing mice, although it did not reach statistical significance. Interestingly, the mean pressure gradient of mitral valve was lower and the deceleration time of mitral valve was prolonged by 25% in junctin-overexpressing mice. Both parameters gave a first indication in vivo of an impaired diastolic ventricular function in this transgenic mouse model.

View this table: [in this window] [in a new window]   Table 3 Echocardiographic and Doppler measurements on junctin-overexpressing (JCN) and wild-type mice (WT)

 
3.4 Contractility in isolated papillary muscles
In the next step, we tested whether the impaired diastolic ventricular function observed in echocardiography may correlate with similar alterations at the level of the intact muscle. Hence, we carried out contractile measurements on isolated papillary muscles. Isolated papillary muscles were stimulated at 0.5 Hz. The contraction amplitude was 14.7±6.0 mN/mm² in wild-type and 5.5±1.5 mN/mm² in junctin-overexpressing muscles (n = 8, P>0.05). The time of relaxation was prolonged by 41% (Fig. 3A). The time of relaxation was 301.9±11.6 ms in junctin-overexpressing and 213.3±9.0 ms in wild-type papillary muscles (n = 8, P<0.05). In addition, the time to peak tension was increased by 15% in junctin-overexpressing compared to wild-type papillary muscles (181.7±8.7 vs. 158.6±5.1 ms, respectively, n = 8, P<0.05). After application of isoproterenol, both time of relaxation and time to peak tension were almost comparable between both groups (Fig. 3B).

View larger version (8K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Effects of junctin overexpression on force of contraction. Force of contraction was measured in right papillary muscles from hearts of wild-type (WT) and junctin-overexpressing (JCN) mice. Representative twitches were superimposed under control conditions (A) and after isoproterenol (10–6 mol/l) exposure (B) in wild-type and junctin-overexpressing muscles. This procedure allows the assessment of effects of isoproterenol on the time course. Note the markedly prolonged mechanical relaxation in junctin-overexpressing muscle preparations under control conditions.

 
3.5 Contractile properties in isolated cardiomyocytes
To determine whether the impaired relaxation in the multicellular papillary muscle corresponds with comparable changes in the single cell, we measured the shortening of cardiomyocytes from wild-type and junctin-overexpressing mice. Cardiomyocytes contracted at a stimulation rate of 0.5 Hz. The maximal cell shortening, calculated as the difference between diastolic and systolic length of the cardiomyocyte, was unchanged between junctin-overexpressing and wild-type mice (Table 4). Consistent with the measurement of time parameters in echocardiography and isolated papillary muscle, the kinetics of cell relaxation were prolonged in junctin-overexpressing mice (Fig. 4A). The time to 90% relaxation was increased by 89% in junctin-overexpressing cardiomyocytes (Table 4).

View this table: [in this window] [in a new window]   Table 4 Contractile parameters, Ca2+ transients, and electrophysiology of isolated cardiomyocytes

 

View larger version (23K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Cell shortening, Ca2+ transients, and effects of caffeine on [Ca]i in cardiomyocytes from wild-type and junctin-overexpressing mouse hearts. Shown are representative tracings of cell shortening (A) and the Indo-1 ratio (B) in single cardiomyocytes from wild-type (WT) and junctin-overexpressing (JCN) mice. The cardiomyocytes were stimulated at 0.5 Hz. There is an increase in the duration of the mechanical relaxation in junctin-overexpressing compared to wild-type cardiomyocytes. In addition, note the marked prolongation of decay of Ca2+ transient in junctin-overexpressing ventricular cells. In addition, Ca2+ transients were monitored in the presence of 10 mM caffeine in wild-type (WT) and junctin-overexpressing (JCN) cardiomyocytes (C). After a 15-s stimulation pause, caffeine was applied for 1 min. The amplitude of caffeine-induced Ca2+ transients was diminished in junctin-overexpressing cells.

 
3.6 Ca²⁺ handling in isolated cardiomyocytes
Is an impaired Ca²⁺ handling the basis for the impaired relaxation in junctin-overexpressing mice? To test this, we measured Indo-1 ratio of diastolic and systolic Ca²⁺, kinetic of Ca²⁺ transients [Ca]_i, and L-type Ca²⁺ channel current (I_Ca) in isolated cardiomyocytes from wild-type and junctin-overexpressing mice. Cardiomyocytes were isolated enzymatically, loaded with Indo-1, and stimulated at 0.5 Hz. Diastolic and systolic Ca²⁺ levels were not different between junctin-overexpressing and wild-type cardiomyocytes (Table 4). This resulted in a comparable peak amplitude of [Ca]_i between both groups (Table 4). Remarkably, the time to 50% decay of [Ca]_i was prolonged by 37% in junctin-overexpressing cardiomyocytes (Fig. 4B, Table 4). To determine the effects of junctin overexpression on SR-Ca²⁺ content, caffeine-induced Ca²⁺ transients were measured. The peak amplitude of [Ca]_i was diminished by 30% under rapid caffeine application in junctin-overexpressing cardiomyocytes (Fig. 4C, Table 4). However, the decay of the caffeine-induced [Ca]_i signal was prolonged by 75% in junctin-overexpressing compared to wild-type cardiomyocytes (Fig. 4C, Table 4). The lower caffeine-induced Ca²⁺ transient peak amplitude implies a reduced SR-Ca²⁺ content, while the slower decline may indicate a reduced Na⁺/Ca²⁺ exchanger function. The activation mode of the ryanodine receptor, which is in part responsible for the SR-Ca²⁺ release, may be modulated by the size and duration of the sarcolemmal Ca²⁺ entry through the L-type Ca²⁺ channel. Hence, we tested whether the impaired kinetics of [Ca]_i in junctin-overexpressing cardiomyocytes were associated with altered characteristics of the L-type Ca²⁺ channel current. The peak current density of I_Ca was not different between junctin-overexpressing and wild-type cardiomyocytes (Table 4). However, the kinetics of inactivation of the L-type Ca²⁺ channel were altered in junctin-overexpressing cardiomyocytes (Fig. 5). Here, we discriminated between a fast (₁) and slow (₂) time constant of inactivation of I_Ca. Both time constants of inactivation of I_Ca were prolonged in cardiomyocytes of junctin-overexpressing hearts (Fig. 5, Table 4). In addition, we used Ba²⁺ as a charge carrier instead of Ca²⁺. Ba²⁺ ions permeating through the L-type Ca²⁺ channel substitute poorly for Ca²⁺ in the Ca²⁺-induced inactivation of the L-type channel, and also are ineffective in inducing Ca²⁺ release from the SR. The peak current density of I_Ba was decreased in junctin-overexpressing cardiomyocytes (Table 4). Consistent with previous results [36], with Ba²⁺ as the charge carrier, the inactivation of I_Ba was prolonged and was best fit by a monoexponential function in mouse cardiomyocytes. The time constant using Ba²⁺, _Ba, was similar to that of the larger time constant of inactivation in the Ca²⁺ containing solution. Nonetheless, _Ba was increased in transgenic cardiomyocytes (Table 4).

View larger version (14K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Inactivation kinetics of ICa. Shown are typical whole-cell L-type Ca2+ channel currents in wild-type (WT) and transgenic (TRD) cardiomyocytes. The time courses of ICa were recorded at a test potential of +10 mV. Note the prolonged inactivation kinetics of ICa.

 

	4. Discussion

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
Cardiac junctin is localized in the junctional SR where it appears to ‘anchor’ calsequestrin to the Ca²⁺ release channel. The functional consequences of this interaction in the heart have not previously been investigated. Here we used a transgenic model of junctin overexpression in the heart to show that overexpression of junctin produces selective down-regulation of other junctional SR proteins, cardiac hypertrophy, and contractile abnormalities, with the most notable effect being on cardiac relaxation parameters.

4.1 Junctin overexpression and hypertrophy
Compared to the hypertrophy observed with overexpression of the associated junctional SR-protein, calsequestrin, which gives a twofold increase in heart weight and progresses rapidly to heart failure and early death [32], the hypertrophy associated with triadin 1 overexpression [20] or the present degree of junctin overexpression (about 10-fold) is relatively benign (16 and 19% increase in heart to body weight ratio at 16–20 weeks, respectively). However, a 29-fold cardiac-specific overexpression of junctin led to a pronounced increase in relative heart weight (increase to 231%) [37] using homozygous mice while we studied heterozygous mice. Here, no increased mortality in 10-fold junctin-overexpressing mice was noted. Junctin overexpression does not appear to yield a phenotype of overt heart failure or early death (this work). However, in 29-fold junctin-overexpressing mice, signs of right heart failure were noted and mortality was greatly increased [37]. Nonetheless, a key indicator of hypertrophy and failure, the elevated ANF mRNA expression, is evident already in 10-fold junctin-overexpressing mice (present paper). It has been shown that an increased diastolic intracellular Ca²⁺ level may act as a hypertrophic signal. This high intracellular Ca²⁺ can activate calcineurin [38] or Ca²⁺/calmodulin-dependent protein kinase II [39,40] leading to cardiac hypertrophy. However, diastolic Ca²⁺ was not increased in the present overexpression model but was decreased in a model increasing junctin 29-fold [37]. The data from the 10-fold junctin-overexpressing mice suggest an impaired Ca²⁺ handling. Indeed, [Ca]_i decline during the twitch was slowed significantly and SR-Ca²⁺ load was reduced. These findings would be consistent with reduced SR-Ca²⁺ATPase function. While there was no change in SERCA2a or phospholamban protein expression, we cannot rule out the possibility that phospholamban is less phosphorylated in the transgenic mice (i.e. which would reduce SR-Ca²⁺ pumping). Another possibility is that there is more maintained Ca²⁺ release via the ryanodine receptor which could extend to an enhanced diastolic SR-Ca²⁺ leak. This might then relate to the major alterations in the expression of junctional SR-proteins in this junctin-overexpressing mouse.

4.2 Changes in the expression level of junctional SR-proteins
Interestingly, transgenic hearts adapted to overexpression of junctin by down-regulating the levels of two other junctional proteins involved in Ca²⁺ release function, the ryanodine receptor and triadin. The level of the other junctional SR-protein, calsequestrin, remained unchanged, as did the levels of the two free SR-proteins, SERCA and its associated regulatory protein phospholamban. A 29-fold degree of junctin overexpression likewise led to a reduction in triadin protein expression while free SR-protein expression remained constant [37]. Similarly, overexpression of triadin 1 led to down-regulation of junctin and the ryanodine receptor while calsequestrin expression, SERCA, and phospholamban expression remained constant. It may be that in attempting to hold the amount of Ca²⁺ release from the junctional SR as close to normal as possible when junctin (or triadin 1) is overexpressed, the SR adapts by down-regulating other junctional proteins, including the ryanodine receptor, triadin or junctin. Calsequestrin may remain unchanged for maintaining intralumenal Ca²⁺ constant, whereas the other junctional SR-proteins may adapt to maintain the net Ca²⁺ release as close to normal as possible. The protein most down-regulated with junctin overexpression, triadin, is the protein most homologous to junctin. Triadin down-regulation (to 20%) is higher in 29-fold junctin-overexpressing mice than in 10-fold junctin-overexpressing mice (to 77%). Moreover, immunoblotting analysis indicated that the protein level of the ryanodine receptor was down-regulated to 68% in 10-fold junctin-overexpressing mouse hearts, whereas the [³H]ryanodine binding assay detected no significant difference. Since ryanodine only binds to open Ca²⁺ release channels, it could be that in junctin-overexpressing hearts, compensatory biochemical changes are occurring allowing the ryanodine receptor to become more easily activatable, thus partially offsetting the substantial decrease in the total level of the ryanodine receptors which are present. Thus, the comparison of both transgenic mouse models (i.e. low and high overexpressors with 10- and 29-fold overexpression of junctin, respectively) allows the assessment of gene dose effects.

The biochemical cause (or causes) of the physiological changes observed in both junctin-overexpressing mouse models are difficult to ascribe directly to over- or underexpression of a single protein, because so many junctional SR-proteins are changing concurrently. What we can conclude is that junctin overexpression in the heart has significant physiological effects on cardiac contractility, which includes aberrations in both net Ca²⁺ uptake and Ca²⁺ release by the SR. Remarkably, the systolic dysfunction occurred only in isolated papillary muscles of junctin-overexpressing mice. However, the contractility was unchanged between both groups in isolated cardiomyocytes and in echocardiography in vivo studies. This apparent discrepancy between the extent of systolic dysfunction in the methods used may be explained by the different physiological conditions of the measurements. While cardiomyocytes exhibit an isotonic contraction, papillary muscles exhibit an isometric contraction in our experimental procedure. Furthermore, the heart contraction in vivo (e.g. detected by echocardiography) is neither isometric not isotonic, but rather auxotonic. Thus, we record contractions under different experimental conditions and this probably explains why differences between wild-type and junctin-overexpressing mice are more prominent in the papillary muscle experiments.

Other effects, such as prolongation of the I_Ca and/or I_Ba kinetics, are probably secondary to impaired Ca²⁺ release from the SR, and not reflective of a direct interaction between junctin and proteins of the sarcolemma such as the L-type Ca²⁺ channel. In 10-fold junctin-overexpressing mice, the peak current through L-type Ca²⁺ channels is unchanged in contrast to an increase in this current in 29-fold junctin-overexpressing mice [37]. For the same reason possibly, bradycardia was noted in 29-fold junctin-overexpressing mice, but was not in the present model. Interestingly, the peak current through L-type Ca²⁺ channels was reduced in 10-fold junctin-overexpressing mice when Ba²⁺ was used as a charge carrier in the extracellular solution. This decrease may indicate altered gating properties of the L-type Ca²⁺ channel in 10-fold junctin-overexpressing mice.

Our data indicate that adult junctin-overexpressing mice apparently escape overt heart failure because other junctional SR-proteins have been down-regulated to allow Ca²⁺ release to remain close to normal at the normal mouse heart rate. To test this hypothesis, we are currently mating junctin-overexpressing mice with triadin 1-overexpressing mice so that forced overexpression of both proteins can occur simultaneously.

4.3 Prolonged relaxation in junctin-overexpressing mice
The relaxation abnormalities are present on all levels which we tested in an integrative approach. This is a novel finding of the present paper. We clearly noted signs of impaired relaxation in echocardiography. The indications of impaired relaxation noted by echocardiography in the intact animal could be due to extracardiac causes like altered afterload. However, relaxation was also prolonged in isolated papillary muscle where the afterload is controlled. Hence, impaired relaxation must be due to cardiac adaptation to junctin overexpression. This conclusion is further supported by contractile abnormalities in isolated electrically stimulated myocytes. The underlying mechanism of the impaired relaxation can be traced to defective Ca²⁺ handling, because cardiomyocytes isolated from junctin-overexpressing mice exhibit prolonged decay of Ca²⁺ transients (Table 4). The slowing of twitch [Ca]_i decline is almost surely due to either slowed SR-Ca²⁺ uptake or prolonged SR-Ca²⁺ release. While we cannot rule either possibility out yet, the prolonged Ca²⁺ release might have a more direct mechanistic connection to the overexpression of junctin. That is, the increased junctin, and decreased ryanodine receptor and triadin may cause an alteration of Ca²⁺ release channel gating. This will require further investigation. In addition, both an apparently slower Na⁺/Ca²⁺ exchanger Ca²⁺ extrusion and/or slower I_Ca inactivation might also contribute to the observed slowing of twitch [Ca]_i decline and relaxation. However, in mouse, the Na⁺/Ca²⁺ exchanger is a very minor player in [Ca]_i decline [41] and the short mouse action potential should also limit the time where I_Ca is relevant.

The present study demonstrates that the overexpression of junctin in the heart is associated with altered expression of junctional SR-proteins, prolonged kinetics of [Ca]_i, and impaired mechanical relaxation.

Time for primary review 28 days.

	Acknowledgements

 
This work was supported by NIH Grant HL-28556 (L.R.J.), the Deutsche Forschungsgemeinschaft and the SFB 556-projects B1 and Z2 (J.N.), the IZKF Münster (L.F.), the ‘Jung-Stiftung für Wissenschaft und Forschung Hamburg’ (B.R.), and a postdoctoral fellowship from the Deutsche Gesellschaft für Kardiologie—Herz- und Kreislaufforschung (U.K.). We thank N. Hinsenhofen for technical assistance.

	References

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 

1. Fabiato A., Fabiato F. Contractions induced by a calcium-triggered release of calcium from the sarcoplasmic reticulum of single skinned cardiac cells. J Physiol (1975) 249:469–495.
2. Sham J.S.K., Cleemann L., Morad M. Functional coupling of Ca²⁺ channels and ryanodine receptors in cardiac myocytes. Proc Natl Acad Sci USA (1995) 92:121–125.
3. Bers D.M. Excitation–contraction coupling and cardiac contractile force. (2001) Boston, MA: Kluwer.
4. Kiriazis H., Kranias E.G. Genetically engineered models with alterations in cardiac membrane calcium-handling proteins. Annu Rev Physiol (2000) 62:321–351.
5. Wagenknecht T., Grassucci R., Frank J., et al. Three-dimensional architecture of the calcium channel/foot structure of sarcoplasmic reticulum. Nature (1989) 338:167–170.
6. Campbell K.P., MacLennan D.H., Jorgensen A.O., Mintzer M.C. Purification and characterization of calsequestrin from canine cardiac sarcoplasmic reticulum and identification of the 53,000 dalton glycoprotein. J Biol Chem (1983) 258:1197–1204.
7. Ikemoto N., Ronjat M., Meszaros L.G., Koshita M. Postulated role of calsequestrin in the regulation of calcium release from sarcoplasmic reticulum. Biochemistry (1989) 28:6764–6771.
8. Shin D.W., Ma J., Kim D.H. The asp-rich region at the carboxyl-terminus of calsequestrin binds to Ca²⁺ and interacts with triadin. FEBS Lett (2000) 486:178–182.
9. Zhang L., Kelly J., Schmeisser G., Kobayashi Y.M., Jones L.R. Complex formation between junctin, triadin, calsequestrin, and the ryanodine receptor. J Biol Chem (1997) 272:23389–23397.
10. Jones L.R., Zhang L., Sanborn K., Jorgensen A.O., Kelley J. Purification, primary structure, and immunological characterization of the 26-kDa calsequestrin binding protein (junctin) from cardiac junctional sarcoplasmic reticulum. J Biol Chem (1995) 270:30787–30796.
11. Kawasaki T., Kasai M. Regulation of calcium channel in sarcoplasmic reticulum by calsequestrin. Biochem Biophys Res Commun (1994) 199:1120–1127.
12. Yamaguchi N., Kawasaki T., Kasai M. DIDS binding 30-kDa protein regulates the calcium release channel in the sarcoplasmic reticulum. Biochem Biophys Res Commun (1995) 210:648–653.
13. Kagari T., Yamaguchi N., Kasai M. Biochemical characterization of calsequestrin-binding 30-kDa protein in sarcoplasmic reticulum of skeletal muscle. Biochem Biophys Res Commun (1996) 227:700–706.
14. Guo W., Campbell K.P. Association of triadin with the ryanodine receptor and calsequestrin in the lumen of the sarcoplasmic reticulum. J Biol Chem (1995) 270:9027–9030.
15. Kobayashi Y.M., Jones L.R. Identification of triadin 1 as the predominant triadin isoform expressed in mammalian myocardium. J Biol Chem (1999) 274:28660–28668.
16. Kobayashi Y.M., Alseikhan B.A., Jones L.R. Localization and characterization of the calsequestrin-binding domain of triadin 1. Evidence for a charged beta-strand in mediating the protein–protein interaction. J Biol Chem (2000) 275:17639–17646.
17. Dinchuk J.E., Henderson N.L., Burn T.C., et al. Aspartyl β-hydroxylase (Asph) and an evolutionarily conserved isoform of Asph missing the catalytic domain share exons with junctin. J Biol Chem (2000) 275:39543–39554.
18. Ohkura M., Furukawa K.I., Fujimori H., et al. Dual regulation of the skeletal muscle ryanodine receptor by triadin and calsequestrin. Biochemistry (1998) 37:12987–12993.
19. Kuniyasu A., Kawano S., Hirayama Y., et al. A new scorpion toxin (BmK-PL) stimulates Ca²⁺-release channel activity of the skeletal-muscle ryanodine receptor by an indirect mechanism. Biochem J (1999) 339:343–350.
20. Kirchhefer U., Neumann J., Baba H.A., et al. Cardiac hypertrophy and impaired relaxation in transgenic mice overexpressing triadin 1. J Biol Chem (2001) 276:4142–4149.
21. Kirchhefer U., Baba H.A., Kobayashi Y.M., et al. Altered function in atrium of transgenic mice overexpressing triadin 1. Am J Physiol (2002) 283:H1334–H1343.
22. Zhang L., Franzini-Armstrong C., Ramesh V., Jones L.R. Structural alterations in cardiac calcium release units resulting from overexpression of junctin. J Mol Cell Cardiol (2001) 33:233–247.
23. Hogan B., Constantini F., Lacy E. Manipulating the mouse embryo: a laboratory manual. (1986) Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
24. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Anal Biochem (1987) 162:156–159.
25. Lowry O.H., Rosebrough N.J., Farr A.L., Randall R.J. Protein measurement with the Folin phenol reagent. J Biol Chem (1951) 193:265–275.
26. Porzio M.A., Pearson A.M. Improved resolution of myofibrillar proteins with sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Biochim Biophys Acta (1977) 490:27–34.
27. Mahony L., Jones L.R. Developmental changes in cardiac sarcoplasmic reticulum in sheep. J Biol Chem (1986) 261:15257–15265.
28. Jones L.R., Field L.J. Residues 2–25 of phospholamban are insufficient to inhibit the Ca²⁺ transport ATPase of cardiac sarcoplasmic reticulum. J Biol Chem (1993) 259:1834–1841.
29. Scatchard G. The attraction of proteins for small molecules and ions. Ann NY Acad Sci (1949) 51:660–672.
30. Boknik P., Neumann J., Kaspareit G., et al. Mechanisms of the contractile effects of levosimendan in the mammalian heart. J Pharmacol Exp Ther (1997) 280:277–283.
31. Gulick J., Subramaniam A., Neumann J., Robbins J. Isolation and characterization of the mouse cardiac myosin heavy chain genes. J Biol Chem (1991) 266:9180–9185.
32. Jones L.R., Suzuki Y.J., Wang W., et al. Regulation of Ca²⁺ signaling in transgenic mouse cardiac myocytes overexpressing calsequestrin. J Clin Invest (1998) 101:1385–1393.
33. Hu K.Y., Huso D.L., Dawson T.M., Bredt D.S., Becker L.C. Nitric oxide synthase in cardiac sarcoplasmic reticulum. Proc Natl Acad Sci USA (1999) 96:657–662.
34. Buchwalow I.B., Schulze W., Karczewski P., et al. Inducible nitric oxide synthase in the myocard. Mol Cell Biochem (2001) 217:73–82.
35. Meissner G. Ryanodine receptor/Ca²⁺ release channels and their regulation by endogenous effectors. Annu Rev Physiol (1994) 56:485–508.
36. Masaki H., Sato Y., Luo W., Kranias E.G., Yatani A. Phospholamban deficiency alters inactivation kinetics of L-type Ca²⁺ channels in mouse ventricular myocytes. Am J Physiol (1997) 272:H606–H612.
37. Hong C.S., Cho M.C., Kwak Y.G., et al. Cardiac remodeling and atrial fibrillation in transgenic mice overexpressing junctin. FASEB J (2002) 16:1310–1312.
38. Molkentin J.D., Lu J.R., Antos C.L., et al. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell (1998) 93:215–228.
39. Ramirez M.T., Zhao X.-L., Schulman H., Brown J.H. The nuclear _B isoform of Ca²⁺/calmodulin-dependent protein kinase II regulates atrial natriuretic factor gene expression in ventricular myocytes. J Biol Chem (1997) 272:31203–31208.
40. Zhang T., Johnson E.N., Gu Y., et al. The cardiac-specific nuclear _B isoform of Ca²⁺/calmodulin-dependent protein kinase II induces hypertrophy and dilated cardiomyopathy associated with increased protein phosphatase 2A activity. J Biol Chem (2002) 277:1261–1267.
41. Li L., Chu G., Kranias E.G., Bers D.M. Cardiac myocyte calcium transport in phospholamban knockout mouse: relaxation and endogenous CaMKII effects. Am J Physiol (1998) 274:H1335–H1347.

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				P. D. Allen Triadin, not essential, but useful J. Physiol., July 1, 2009; 587(13): 3123 - 3124. [Full Text] [PDF]

				T. J. Pritchard and E. G. Kranias Junctin and the histidine-rich Ca2+ binding protein: potential roles in heart failure and arrhythmogenesis J. Physiol., July 1, 2009; 587(13): 3125 - 3133. [Abstract] [Full Text] [PDF]

				J.-B. Shen, R. Shutt, M. Agosto, A. Pappano, and B. T. Liang Reversal of cardiac myocyte dysfunction as a unique mechanism of rescue by P2X4 receptors in cardiomyopathy Am J Physiol Heart Circ Physiol, April 1, 2009; 296(4): H1089 - H1095. [Abstract] [Full Text] [PDF]

				S. Grote-Wessels, H. A. Baba, P. Boknik, A. El-Armouche, L. Fabritz, H.-J. Gillmann, D. Kucerova, M. Matus, F. U. Muller, J. Neumann, et al. Inhibition of protein phosphatase 1 by inhibitor-2 exacerbates progression of cardiac failure in a model with pressure overload Cardiovasc Res, August 1, 2008; 79(3): 464 - 471. [Abstract] [Full Text] [PDF]

				S. Gyorke and D. Terentyev Modulation of ryanodine receptor by luminal calcium and accessory proteins in health and cardiac disease Cardiovasc Res, January 15, 2008; 77(2): 245 - 255. [Abstract] [Full Text] [PDF]

				U. Kirchhefer, J. Klimas, H. A. Baba, I. B. Buchwalow, L. Fabritz, M. Huls, M. Matus, F. U. Muller, W. Schmitz, and J. Neumann Triadin is a critical determinant of cellular Ca cycling and contractility in the heart Am J Physiol Heart Circ Physiol, November 1, 2007; 293(5): H3165 - H3174. [Abstract] [Full Text] [PDF]

				U. Gergs, T. Berndt, J. Buskase, L. R. Jones, U. Kirchhefer, F. U. Muller, K.-D. Schluter, W. Schmitz, and J. Neumann On the role of junctin in cardiac Ca2+ handling, contractility, and heart failure Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H728 - H734. [Abstract] [Full Text] [PDF]

				Q. Yuan, G.-C. Fan, M. Dong, B. Altschafl, A. Diwan, X. Ren, H. H. Hahn, W. Zhao, J. R. Waggoner, L. R. Jones, et al. Sarcoplasmic Reticulum Calcium Overloading in Junctin Deficiency Enhances Cardiac Contractility but Increases Ventricular Automaticity Circulation, January 23, 2007; 115(3): 300 - 309. [Abstract] [Full Text] [PDF]

				E. J. Jaehnig, A. B. Heidt, S. B. Greene, I. Cornelissen, and B. L. Black Increased Susceptibility to Isoproterenol-Induced Cardiac Hypertrophy and Impaired Weight Gain in Mice Lacking the Histidine-Rich Calcium-Binding Protein Mol. Cell. Biol., December 15, 2006; 26(24): 9315 - 9326. [Abstract] [Full Text] [PDF]

				S. S. Rezgui, S. Vassilopoulos, J. Brocard, J. C. Platel, A. Bouron, C. Arnoult, S. Oddoux, L. Garcia, M. De Waard, and I. Marty Triadin (Trisk 95) Overexpression Blocks Excitation-Contraction Coupling in Rat Skeletal Myotubes J. Biol. Chem., November 25, 2005; 280(47): 39302 - 39308. [Abstract] [Full Text] [PDF]

				U. Kirchhefer, H. A. Baba, P. Boknik, K. M. Breeden, N. Mavila, N. Bruchert, I. Justus, M. Matus, W. Schmitz, A. A. DePaoli-Roach, et al. Enhanced cardiac function in mice overexpressing protein phosphatase Inhibitor-2 Cardiovasc Res, October 1, 2005; 68(1): 98 - 108. [Abstract] [Full Text] [PDF]

				D. Terentyev, S. E. Cala, T. D. Houle, S. Viatchenko-Karpinski, I. Gyorke, R. Terentyeva, S. C. Williams, and S. Gyorke Triadin Overexpression Stimulates Excitation-Contraction Coupling and Increases Predisposition to Cellular Arrhythmia in Cardiac Myocytes Circ. Res., April 1, 2005; 96(6): 651 - 658. [Abstract] [Full Text] [PDF]

				U. Kirchhefer, H. A. Baba, G. Hanske, L. R. Jones, P. Kirchhof, W. Schmitz, and J. Neumann Age-dependent biochemical and contractile properties in atrium of transgenic mice overexpressing junctin Am J Physiol Heart Circ Physiol, November 1, 2004; 287(5): H2216 - H2225. [Abstract] [Full Text] [PDF]

				G.-C. Fan, K. N. Gregory, W. Zhao, W. J. Park, and E. G. Kranias Regulation of myocardial function by histidine-rich, calcium-binding protein Am J Physiol Heart Circ Physiol, October 1, 2004; 287(4): H1705 - H1711. [Abstract] [Full Text] [PDF]

				V. E. Bondarenko, G. P. Szigeti, G. C. L. Bett, S.-J. Kim, and R. L. Rasmusson Computer model of action potential of mouse ventricular myocytes Am J Physiol Heart Circ Physiol, September 1, 2004; 287(3): H1378 - H1403. [Abstract] [Full Text] [PDF]

				U. Kirchhefer, L. R Jones, F. Begrow, P. Boknik, L. Hein, M. J Lohse, B. Riemann, W. Schmitz, J. Stypmann, and J. Neumann Transgenic triadin 1 overexpression alters SR Ca2+ handling and leads to a blunted contractile response to {beta}-adrenergic agonists Cardiovasc Res, April 1, 2004; 62(1): 122 - 134. [Abstract] [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Disclaimer Google Scholar Articles by Kirchhefer, U. Articles by Jones, L. R. Search for Related Content PubMed PubMed Citation Articles by Kirchhefer, U. Articles by Jones, L. R. Social Bookmarking          What's this?

Online ISSN 1755-3245 - Print ISSN 0008-6363

Copyright © 2009 European Society of Cardiology

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics