AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 293: H728-H734, 2007. First published March 30, 2007; doi:10.1152/ajpheart.01187.2006
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On the role of junctin in cardiac Ca2+ handling, contractility, and heart failure

Ulrich Gergs,1 Tobias Berndt,3 Jan Buskase,2 Larry R. Jones,4 Uwe Kirchhefer,2 Frank U. Müller,2 Klaus-Dieter Schlüter,3 Wilhelm Schmitz,2 and Joachim Neumann1

1Institut für Pharmakologie und Toxikologie, Martin-Luther-Universität Halle-Wittenberg, Halle (Saale), Germany; 2Institut für Pharmakologie und Toxikologie, Westfälische Wilhelms-Universität, Münster, Germany; 3Physiologisches Institut, Justus-Liebig-Universität, Giebetaen, Germany; and 4Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis, Indiana

Submitted 30 October 2006 ; accepted in final form 29 March 2007


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Junctin is a transmembrane protein located at the cardiac junctional sarcoplasmic reticulum (SR) and forms a quaternary complex with the Ca2+ release channel, triadin and calsequestrin. Impaired protein interactions within this complex may alter the Ca2+ sensitivity of the Ca2+ release channel and may lead to cardiac dysfunction, including hypertrophy, depressed contractility, and abnormal Ca2+ transients. To study the expression of junctin and, for comparison, triadin, in heart failure, we measured the levels of these proteins in SR from normal and failing human hearts. Junctin was below our level of detection in SR membranes from failing human hearts, and triadin was downregulated by 22%. To better understand the role of junctin in the regulation of Ca2+ homeostasis and contraction of cardiac myocytes, we used an adenoviral approach to overexpress junctin in isolated rat cardiac myocytes. A recombinant adenovirus encoding the green fluorescent protein served as a control. Infection of myocytes with the junctin-expressing virus resulted in an increased RNA and protein expression of junctin. Ca2+ transients showed a decreased maximum Ca2+ amplitude, and contractility of myocytes was depressed. Our results demonstrate that an increased expression of junctin is associated with an impaired Ca2+ homeostasis. Downregulation of junctin in human heart failure may thus be a compensatory mechanism.

junctin; adenovirus; cardiac myocytes; calcium transient; sarcoplasmic reticulum

CALCIUM, THE KEY REGULATOR of cardiac excitation-contraction coupling, enters the cardiac myocyte through the L-type Ca2+ channel, and triggers the release of Ca2+ from the sarcoplasmic reticulum (SR) via activation of the ryanodine receptor (RyR). The ensuing increase in intracellular Ca2+ concentration initiates muscle contraction (1, 2). For relaxation, Ca2+ is removed from the cytosol by the action of SR Ca2+-ATPase (SERCA) and by the Na+/Ca2+ exchanger (NCX) in the sarcolemma. Alterations in intracellular Ca2+ homeostasis appear to play a crucial role in contractile dysfunction in heart failure. At least three mechanisms can contribute to a reduced SR Ca2+ load, resulting in contractile dysfunction. These are a reduced SERCA activity, leading to less Ca2+ being pumped into the SR lumen; enhanced NCX function causing Ca2+ loss at the plasma membrane; and an augmented SR Ca2+ leak from the SR lumen, as the result of defective regulation of RyRs (3, 7). In the present study, we focused on possible abnormalities in the SR. At the luminal site of RyR, calsequestrin (CSQ) (26) a Ca2+-binding protein with high capacity and low affinity for Ca2+ is anchored to the RyR by the SR transmembrane proteins triadin 1 (TRD) (22) and junctin (JCN) (12). TRD and JCN are homologous proteins constituted of single transmembrane domains and highly charged carboxy termini within the SR lumen (20). RyR, CSQ, TRD, and JCN probably form a quaternary complex in the junctional SR (12, 21, 36). CSQ, TRD, and JCN may mediate the Ca2+ sensitivity of RyR (5, 12, 22, 33). The exact role of JCN is still unknown. It may act like TRD, as a Ca2+ sensor per se, or mediate the interaction between RyR and CSQ (12, 21). This issue was addressed by transgenic approaches in the past. Cardiac-specific overexpression of either CSQ, TRD, or JCN in transgenic mice resulted in increased expression of the transgene but also to reduced expression of other proteins in the SR. (11, 17, 18, 35). A more rigorous understanding of the cross talk between these proteins of the junctional SR might lead to novel therapies for the treatment of human heart failure.

Here, we demonstrate that expression of JCN is reduced below our limit of detection in human heart failure. To study the function of JCN in more detail, we used rat cardiac myocytes for adenoviral overexpression of JCN and found that an increased expression of JCN can lead to decreased Ca2+ transients and depressed contractility. The advantage of the model used here lies in the apparent lack of counterregulatory expressional changes, which hamper the interpretation of previous studies in mice overexpressing the transgene. It is speculated by extrapolation that reduced levels of JCN in failing human hearts are intended to maintain Ca2+ high in the SR and are thus a compensatory mechanism.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
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Human cardiac tissue. Samples were taken from left ventricles of failing human hearts that were explanted in the course of heart transplantation as described before (28). Nonfailing hearts were obtained from prospective organ donors whose hearts could not be used. The study's protocol was approved by the local ethics committee and patients gave written informed consent.

Recombinant adenoviruses. A recombinant adenovirus carrying the canine JCN gene (Ad-JCN) was generated and amplified, as described elsewhere (9). As shuttle vector pAdTrack-cytomegalovirus (CMV) containing the reporter gene green fluorescent protein (GFP) was used. Both, GFP and the gene of interest were expressed under control of a separate CMV promoter. As an adenoviral backbone plasmid, we used pAdEasy-1 containing the Ad5 genome with deletion of the E1 and E3 genes. Briefly, the cDNA of JCN was amplified using standard RT-PCR methodology and cloned into the shuttle vector. The resultant plasmid was linearized by PmeI digestion and cotransformed into Escherichia coli cells with the adenoviral backbone plasmid for homologous recombination. Finally, the recombinant adenoviral vector DNA was digested with PacI and transfected into the adenovirus packaging cell line HEK293. A GFP-only virus (Ad-Control) was used as a control adenovirus. After three rounds of amplification, the viral titers, determined using the cytopathic effect of the viruses, were high enough to use for gene transfer experiments in cultured cells.

Neonatal rat cardiac myocytes. Myocytes from hearts of 1- to 3-day-old Wistar rats were isolated using the neonatal cardiomyocyte isolation system purchased from Worthington Biochemical (Cell Systems, Katharinen, Germany) following the instructions provided by the manufacturer. In brief, cells were dissociated from ventricles by incubation with trypsin and collagenase. The dissociated cells were resuspended in culture medium (DMEM supplemented with 10% horse serum, 2% fetal calf serum, 2 mM L-glutamine, and 100 units/ml of both penicillin and streptomycin) and preplated for 30 min to reduce fibroblast contamination. The unattached cells were transferred to collagen-coated plates with a final density of 125,000 cells per cm2 and cultured at 37°C in a humidified incubator containing 5% CO2. Every 2 days, the culture medium was changed. Two days after initial plating, myocytes were infected with adenoviruses at a multiplicity of infection (MOI) of 100 and were cultured an additional 2 days before beginning the experiments.

Adult rat cardiac myocytes. Ventricular heart muscle cells were isolated from 200- to 250-g male Wistar rats. Hearts were excised in deep ether anesthesia, rapidly transferred to ice-cold saline, and mounted on the cannula of a Langendorff perfusion system. Heart perfusion and subsequent steps were all performed at 37°C. First, hearts were perfused in a noncirculating manner for 5 min at 10 ml/min (composition of perfusate in mM: 110 NaCl, 1.2 KH2PO4, 2.6 KCl, 1.2 MgSO4, 25 NaHCO3, 11 glucose, gassed with 5% CO2-95% O2). Thereafter, perfusion was continued by recirculation of 50 ml of the above perfusate supplemented with 0.06% (wt/vol) crude collagenase and 25 µM CaCl2 at 5 ml/min. After 30 min, ventricular tissue was minced and incubated for 20 min in recirculating medium with 1% (wt/vol) bovine serum albumin under 5% CO2-95% O2. By gentle pipetting, cells were released from tissue chunks. The resulting cell suspension was filtered through a 200-µm-nylon mesh. Twice, the filtered material was washed by centrifugation (3 min, 25 g) and resuspended in the above perfusate, in which the concentration of CaCl2 was stepwise increased to 0.2 and 0.5 mM. After another centrifugation (3 min, 25 g), the cells in the pellet were suspended in serum-free culture medium (medium 199 with Earle's salts, 5 mM creatine, 2 mM L-carnitine, 5 mM taurine, 100 IU/ml penicillin, and 100 µg/ml streptomycin) and plated at a density of 7 x 104 elongated cells/35-mm culture dish (Falcon, type 3001). The culture dishes had been preincubated overnight with 4% FCS in medium 199. Two hours after plating, cultures were washed with modified Tyrode solution (composition in mM: 125 NaCl, 1.2 KH2PO4, 2.6 KCl, 1.2 MgSO4, 1 CaCl2, 10 glucose, 10 HEPES, pH 7.4) to remove round and nonattached cells, in which cell contractions were analyzed. Myocytes were infected with adenoviruses at a MOI of 100 and cultured an additional 2 days before beginning with the experiments.

Northern blot analysis. Total RNA from adenoviral infected or noninfected control cells was isolated using the RNeasy-Kit (QIAGEN, Cologne, Germany) according to the manufacturers instructions. For Northern blot analysis, 10 µg of total RNA were separated by electrophoresis and transferred to nylon membranes, and hybridization was performed with a [32P] cDNA of JCN.

Preparation of cell lysates and tissue homogenates. Cultured cells, adenovirus infected or noninfected, were washed once with PBS at 4°C and collected in lysis buffer (0.2 ml per 3.5 cm dish) containing 10 mM histidine (pH 7.4), 250 mM saccharose, and 1% (wt/vol) SDS. Remaining adenoviruses were inactivated by boiling the lysate for 5 min. Finally, solubilization was completed by sonication for 30 s (VirSonic, Virtus, NY). The lysate was cleared by centrifugation for 10 min at 14,000 g. Protein concentrations were measured according to the method of Lowry et al. (24). Frozen tissue samples were pulverized in a mortar precooled in liquid nitrogen. The following steps were carried out at 4°C. Ten volumes of homogenization buffer containing 10 mM histidine (pH 7.4), and 250 mM saccharose were added to the frozen, pulverized tissue. The tissue was then homogenized three times for 30 s each with a Polytron PT-10 (Kinematica, Lucerne, Switzerland), and the homogenate was cleared by centrifugation for 10 min at 14,000 g. Protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad, Munich, Germany).

Western blot analysis. For Western blot analysis, cell lysates or tissue homogenates were prepared, and 4x strength SDS buffer containing 250 mM Tris·HCl (pH 6.8), 20% (wt/vol) SDS, 40% (vol/vol) glycerol, 1.2% (wt/vol) dl-dithiothreitol, and 0.004% (wt/vol) bromophenol blue was added. The samples were solubilized for 10 min at 95°C. Aliquots of 50–100 µg protein were loaded per lane, and gels were run using 10% polyacrylamide separating gels. Proteins were electrophoretically transferred to nitrocellulose membranes in 50 mM sodium phosphate buffer (pH 7.4) 180 min at 1.5 A at 4°C. Then, membranes were treated with Tris-buffered saline containing 5.0% nonfat dry milk powder and 0.1% Tween 20 for 120 min at room temperature followed by incubation with primary antibodies overnight at 4°C and subsequently with 125I-labeled protein A. Alternatively, alkaline phosphatase-labeled secondary antibodies were used, and bands were detected using enhanced chemofluorescence (Amersham Biosciences, Piscataway, NJ). Fluorescent bands as well as 125I were visualized in a STORM PhosphorImager and quantified using the ImageQuant software (Amersham Biosciences). The following primary antibodies were used: polyclonal rabbit anti calsequestrin, polyclonal rabbit anti triadin, monoclonal mouse antijunctin, and polyclonal rabbit antijunctin, which have been all published (22, 25, 31, 35).

Ca2+ transients of isolated rat cardiomyocytes. Ventricular myocytes from neonatal or adult rats were prepared and infected with adenoviruses, as described above. Measurement of Ca2+ transients was performed as described previously (17). Briefly, cardiomyocytes were labeled with the Ca2+-binding fluorescence dye Indo-1/AM. After excitation at 365 nm, the emitted fluorescence was recorded at 405 and 495 nm. The ratio of fluorescence at the two wavelengths was used as an index of cytosolic Ca2+ concentration ([Ca]i). To assess the SR-Ca2+ load, rapid application of 10 mM caffeine was used as an established tool (18). The peak amplitude of basal and caffeine-induced Ca2+ transients was determined.

Contraction of isolated adult cardiomyocytes. Cell contractions were performed at room temperature and analyzed via a cell-edge detection system (23). Cells were stimulated via two AgCl electrodes with biphasic electrical stimuli composed of two equal but opposite rectangular 50-V stimuli of 0.5-ms duration. Each cell was stimulated at 1, 0.5, and 2 Hz for 1 min. Every 15 s, the next five contractions were averaged. From these four measurements at a given frequency, the means were used to define cell shortening of a given cell. Cell lengths were measured at a rate of 500 Hz via a line camera.

Statistics. Data are presented as means ± SE. Comparisons between two groups were evaluated using Student's t-test and one-way ANOVA followed by Bonferroni's test was used for multiple-group comparisons. A value of P < 0.05 was considered statistically significant.


   RESULTS
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DISCUSSION
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JCN expression in the failing human heart. Heart samples from patients suffering from dilated cardiomyopathy (DCM; n = 7), and therefore undergoing heart transplantation, were analyzed by Western blot analysis for JCN expression. Nonfailing (NF; n = 7) controls were obtained from hearts unsuitable for transplantation. The antibody against JCN used here has been raised against canine cardiac JCN but cross-reacts with cardiac JCN of other species like mouse, rat, and man (22, 25, 31, 35). First, linearity of the signals detected by Western blot analysis of samples from NF hearts was confirmed (Fig. 1A). The expression of JCN was almost lacking in failing human hearts (Fig. 1C). For comparison, quantification of NF and DCM Western blots revealed a decrease of TRD in DCM hearts by 22% compared with NF hearts (Fig. 1B). As an internal control for protein loading and myocyte content of the samples, expression of CSQ was analyzed. Here, no differences in samples from NF and DCM hearts were detected, in agreement with the literature (6, 27) (Fig. 1C).


Figure 1
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  		Fig. 1. Triadin (TRD), junctin (JCN), and calsequestrin (CSQ) expression in the failing human heart. Heart tissue samples from nonfailing (NF) and failing (F, dilated cardiomyopathy) human hearts were subjected to Western blot analysis. A: expression analysis of TRD ({Psi}, glycosylated form of TRD) and JCN in NF heart samples with increasing protein concentrations was performed to ensure linearity of the assay. B: quantification of TRD expression in samples of NF and failing hearts revealed a decrease of TRD in failing hearts by 22%. A representative blot of TRD is shown. C: three representative Western blots of JCN and CSQ in individual samples from NF and failing hearts. JCN expression in failing hearts (n = 7) was below the detection limit of the methods used here. Therefore, quantification of the decrease was not possible. Unchanged levels of calsequestrin (CSQ) demonstrated that the myocyte content was not different between NF and failing hearts. *P < 0.05 vs. NF.

 
Adenovirus-mediated overexpression of JCN. For the basal characterization of the Ad-JCN, neonatal rat cardiac myocytes were used. This was done because neonatal cells are easy to isolate and handle and are usually good targets for adenoviral infection. Each experiment was repeated at least three times with independent cell preparations. Infection of neonatal cells with a MOI of 100 for 2 days resulted in a complete transfection of cells assessed by GFP fluorescence (not shown). Southern blot analysis of genomic DNA from neonatal cells with a probe against the adenovirus backbone revealed a comparable infection rate in Ad-Control- and Ad-JCN-infected cells (Fig. 2A). A high mRNA expression of canine JCN was detected by Northern blot analysis in neonatal cardiac myocytes infected with Ad-JCN but not with Ad-Control (Fig. 2B). The effect of adenovirus infection on the protein expression in neonatal cells was analyzed by Western blot analysis. Neonatal rat cardiac myocytes were infected with Ad-JCN at different MOI for 2 days. The protein expression was increased in parallel with virus concentrations (Fig. 2C). JCN was only detectable in infected neonatal cardiac myocytes. Similarly, a very low expression of JCN in neonatal rat hearts has been demonstrated recently by Jung et al. (13). However, the SR of neonatal myocytes is immature and poorly developed. Hence, to be able to do meaningful extrapolations for the adult human heart, subsequent studies were done on adult rat myocytes. Infection of adult rat cardiac myocytes with Ad-Control or Ad-JCN at a MOI of 100 for 1, 2, and 3 days resulted in an increased expression of JCN (Fig. 3A). In adult myocytes the endogenous protein was detectable as shown in Ad-Control-infected cells (Fig. 3A). Because canine JCN (210 amino acids; used in our adenovirus) has a higher molecular weight than rat JCN (207 amino acids), endogenous and transgenic JCN can be separated by Western blot analysis in infected rat myocytes (35). Quantification of the specific protein bands revealed a twofold overexpression of JCN (canine and rat JCN together) at day 2 compared with Ad-Control-infected cells (Fig. 3B). The level of endogenous JCN was not altered (P > 0.05). For comparison, protein expression of SERCA, CSQ, and TRD were analyzed in noninfected, Ad-Control, and Ad-JCN-infected adult myocytes (Fig. 3A). Quantification of the protein bands after 2 days of infection is shown (Fig. 3B). In the context of the present work, it is important to note that overexpression of JCN did not influence the expression of other SR-proteins studied here. This constitutes, in our view, an advantage compared with transgenic animal studies, as mentioned in the introduction. The biochemical results presented above were used to define the experimental conditions for the subsequent physiological studies. That is, all physiological experiments were carried out with cardiac myocytes infected for 2 days at an MOI of 100. Of note, cells were damaged histologically after 3 days of infection and therefore were not used in subsequent physiological experiments.


Figure 2
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  		Fig. 2. Adenoviral overexpression of JCN in neonatal rat cardiac myocytes. Isolated neonatal rat cardiac myocytes were infected [multiplicity of infection (MOI) = 100] with Ad-Control or Ad-JCN for 48 h. A: Southern blot analysis of genomic DNA using a probe against the adenovirus backbone shows a comparable infection rate in each experiment (the arrow indicates the adenovirus DNA). DNA from noninfected cells was used as negative control and purified adenovirus DNA (plasmid) as a positive control. B: Northern blot analysis of total RNA using a cDNA-probe against JCN revealed a high mRNA expression of the transgene in neonatal rat cells infected with the Ad-JCN. Ethidium bromide staining of the gel served as loading control. C: cardiac myocytes were infected with Ad-JCN at different MOI for 48 h. Western blot analysis shows increasing protein levels of JCN in infected neonatal cardiomyocytes with increasing virus concentrations.

 

Figure 3
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  		Fig. 3. Time-dependent effect of adenovirus infection on protein expression in adult rat cardiac myocytes. Each cell preparation (n = 3) was split to get enough samples for all conditions. A: Western blot analysis shows protein expression of SR Ca2+-ATPase (SERCA), CSQ, TRD, and JCN in adult rat cardiac myocytes either noninfected (Non-inf.) or infected with Ad-Control or Ad-JCN adenoviruses (MOI = 100) for 1, 2, and 3 days. The JCN antibody used in this experiment recognized the overexpressed canine protein, as well as the endogenous rat homolog. B: quantification of protein expression 2 days after infection of myocytes with Ad-JCN relative to Ad-Control-infected cells. *P < 0.05 vs. Ad-Control.

 
Ca2+ transients in adenovirus-infected cardiomyocytes. Isolated adult rat cardiac myocytes were infected with Ad-Control or Ad-JCN. Ca2+ transients were measured with a pacing rate of 2 Hz in adult myocytes. There was no difference between noninfected and Ad-Control-infected cells (P > 0.05). Representative traces of Ca2+ transients in Ad-Control- and Ad-JCN-infected adult myocytes are shown (Fig. 4A). Overexpression of JCN resulted in a decrease in the peak amplitude for Ca2+ transients relative to Ad-Control-infected cells by 35% [adult myocytes: 0.26 ± 0.02 (Ad-Control) vs. 0.17 ± 0.01 (Ad-JCN), P < 0.05]. Diastolic Ca2+ was not altered in JCN-overexpressing myocytes (P > 0.05). To determine the effect of JCN overexpression on SR-Ca2+ content, caffeine-induced Ca2+ transients were measured (Fig. 4B). In Ad-JCN-infected adult cells, caffeine-induced Ca2+ transients were diminished compared with Ad-Control-infected myocytes by 33% indicative for a reduced SR-Ca2+ content (P < 0.05). Next, we wanted to know whether reduced Ca2+ transients translated into decreased contractility.


Figure 4
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  		Fig. 4. Effect of adenoviral JCN overexpression on Ca2+ transients in cardiac myocytes. Isolated adult rat cardiac myocytes were infected (MOI = 100) with Ad-Control or Ad-JCN for 48 h and Ca2+ transients were measured at 2.0 Hz stimulation rate. A: representative traces of Ca2+ transients in Ad-Control and Ad-JCN-infected myocytes. B: repre sentative traces of caffeine-induced Ca2+ transients.

 
Cell shortening of adenovirus-infected cardiomyocytes. Isolated adult rat cardiac myocytes were infected with Ad-Control or Ad-JCN. Cardiomyocytes were electrically paced at 0.5, 1.0, and 2.0 Hz, and cell shortening was monitored via a cell-edge detection system that allowed us to analyze fractional shortening and the dynamics of contraction. Representative traces are shown in Fig. 5. The overall characteristics of the twitch performance of isolated adult rat ventricular cardiomyocytes have been described before (23). All infected cardiomyocytes showed a drop in contractile responsiveness from 0.5 to 2.0 Hz (Fig. 5). Fractional shortening of Ad-JCN-infected cells was not different from Ad-Control-infected cells. However, the maximum velocity of contraction was slower in cardiac myocytes overexpressing JCN compared with Ad-Control-infected cells (Table 1), and the velocity of relaxation was reduced in Ad-JCN-infected cells at 2.0 Hz (Table 1).


Figure 5
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  		Fig. 5. Effect of adenoviral JCN overexpression on cell shortening of cardiomyocytes. Adult rat cardiac myocytes were infected (MOI = 100) for 48 h either with a control virus (Ad-Control) or with an adenovirus leading to overexpression of JCN. Data are frequency dependent cell shortening normalized to diastolic cell length (dL/L). Original registrations of cell shortening at 0.5, 1.0, and 2.0 Hz stimulation rate are shown.

 

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  		Table 1. Frequency dependence of velocities of contraction and relaxation in adenovirus-infected adult rat cardiac myocytes

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we demonstrated a reduced expression of JCN in human heart failure. To gain more insight into the function of JCN, we used an adenoviral approach to overexpress JCN in isolated rat cardiac myocytes and found a decreased SR Ca2+ transient, a decreased Ca2+ content of the SR, and an attenuated contractility of cardiac myocytes.

In failing human hearts, SR Ca2+ uptake is defective and/or SR Ca2+ release is impaired. Many researchers who have examined the possible mechanism(s) of heart failure have reported a reduced expression and/or an impaired function of SERCA2a, a protein of the free SR (6, 8). Less is known about a potential role of junctional SR proteins in the regulation of SR Ca2+ release in human heart failure. Expression of cardiac CSQ appears to be under tight control, as expression levels remain unchanged in human heart failure (27). A loss of CSQ protein or function due to mutations in the cardiac CSQ gene (CASQ2) was suggested to cause catecholamine-induced ventricular tachycardia (30). This kind of arrhythmia was also observed in mice lacking CASQ2, although, very surprisingly, Ca2+ transients remained largely unaltered in myocytes from CSQ knockout mice (19).

Here, we have shown for the first time a decreased protein expression of JCN (and TRD) in human heart failure. This downregulation was not the result of the loss of muscle cells in the failing hearts, as CSQ expression remained unchanged. However, at present, it is not clear whether the reduced expression of JCN (or TRD) causes heart failure in man or whether downregulation is adaptative to improve Ca2+ handling. TRD may inhibit the Ca2+ release in vivo (15, 16). Alternatively, TRD and/or JCN can increase RyR channel open probability (5). For TRD this was shown in vitro and in isolated cardiac myocytes. Increased expression of TRD in cardiac cells resulted in reduced Ca2+ transients and increased Ca2+ spark frequencies (32). Therefore, overexpression of TRD possibly makes the SR more leaky for Ca2+, leading finally to a loss of cytosolic Ca2+ and hence reduced contractility. Downregulation of JCN in failing human hearts may protect against Ca2+ loss from the SR and may be beneficial for cardiac function. It is possible that downregulation of JCN is an adaptation of the heart to, for example, chronic beta-adrenergic stimulation. This hypothesis is in accordance with a transgenic mouse model of heart failure in which cardiac-specific overexpression of the beta1-adrenoceptor was accompanied by a decrease in the expression of JCN (4). In addition, incorrect junctional SR trafficking and Ca2+ release complex assembly, leading to impaired SR Ca2+ release, may contribute to heart failure (14, 29). Because of these findings in human failing hearts and other models of heart failure, we hypothesize that an enhanced level of JCN relative to the RyR may result in a decreased SR Ca2+ release and depressed contractility. In this study, we present data from JCN-overexpressing rat cardiac myocytes, which support this hypothesis.

Previous studies with constitutive cardiac-directed overexpression of JCN in transgenic mice resulted in various degrees of cardiac hypertrophy, depressed contractility, altered gene expression, and impaired relaxation (10, 18, 35). In hearts of mice with JCN overexpression, protein levels of RyR and TRD were decreased (18, 35). The cardiac phenotype was gene-dose dependent, as demonstrated by transgenic mouse models either with 10- or 30-fold overexpression of canine JCN. In cardiac myocytes with 10-fold overexpression of JCN, morphological changes were visible: the association of SR and T tubules was facilitated and the packing of CSQ was affected (35). With even higher overexpression (30-fold), the phenotype was much more pronounced: transgenic mice exhibited cardiac hypertrophy, bradycardia, atrial fibrillation, and fibrosis (10). It is plausible that these observations not only reflect the primary effects of the JCN overexpression but also secondary effects, as a consequence of hypertrophy and/or compensatory alterations of gene expression (like reduced levels of triadin and RYR). In the present study, we used an adenoviral approach to overexpress JCN for a short time in isolated adult cardiac myocytes to minimize any adaptive changes and to dissect the molecular function of this SR protein better. In the transgenic animal models mentioned above, a common observation was the altered expression profile of junctional SR proteins. This regulation was suggested to maintain the SR Ca2+ homeostasis (11, 17, 35). In contrast, twofold overexpression of JCN, presented here after two days of Ad-JCN infection, had no influence on the levels of other SR proteins like TRD or CSQ (Fig. 3). An important caveat is that we cannot rule out compensatory changes of RyR expression in our adenoviral model: RyR expression was below our level of detection.

Here, cardiomyocytes infected with Ad-JCN exhibited decreased Ca2+ transients and SR Ca2+ content, as well as reduced velocities of contraction and relaxation. Adenoviral overexpression of TRD reduced the amplitude of Ca2+ transients, and the open probability of RyR channels was increased in this model leading to a reduced SR Ca2+ content (32). In contrast to our results, cardiac myocytes from transgenic mice overexpressing JCN showed unchanged peak amplitudes of Ca2+ transients, but the SR-Ca2+ content was also reduced (18). As mentioned above, probably compensatory mechanisms like downregulation of other SR proteins occur in transgenic mice to compensate any alterations caused by overexpression of the transgene. This may explain differences in Ca2+ handling between transgenic animal models and the cell culture model that was used here. Another more general problem of all overexpression models may be the subcellular targeting of the transgene. Incorrect targeting may result in nonspecific side effects, especially if the expressional level is high. But as JCN is thought to be active only in a stable quarternary complex together with TRD, CSQ, and RyR and because of the low overexpression level (2-fold) presented here, nonspecific effects of JCN overexpression seem unlikely, but clearly cannot be excluded.

In summary, it is known that a complex of CSQ, TRD, and/or JCN probably confers luminal Ca2+ sensitivity to RyR (5). All studies published so far have the finding in common that genetically induced changes of either CSQ, TRD, or JCN levels within the SR-Ca2+ release complex led to impaired Ca2+ homeostasis. The molecular mechanisms seem to be different for each model. We demonstrate a reduced expression of JCN (and TRD) in human heart failure, conceivably as an adaptation to a lower SR-Ca2+ content or to compensate an enhanced SR Ca2+ leak. Using adenoviral gene transfer, we have demonstrated the influence of an altered junctional protein composition on SR Ca2+ content, cytosolic Ca2+, and contractile function. While the present manuscript was under review, the phenotype of a junctin knockout was published (34). There, lack of junctin increased Ca2+ transients. Their data are, hence, complementary to our present results. It is tempting to speculate that the lack of junctin in heart failure is a compensatory mechanism in man.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
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This work was supported by the Deutsche Forschungsgemeinschaft and by the Roux Program of the medical faculty of the University of Halle-Wittenberg (Halle, Germany).


   ACKNOWLEDGMENTS
 
We thank Nadine Lorenz and Nicole Hinsenhofen for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: U. Gergs, Institut für Pharmakologie und Toxikologie, Martin-Luther-Universität Halle-Wittenberg, Magdeburger Str. 4, 06112 Halle (Saale), Germany (e-mail: ulrich.gergs{at}medizin.uni-halle.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
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