Cardiovascular Research

Cardiovascular Research 2005 65(1):177-186; doi:10.1016/j.cardiores.2004.08.012

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Copyright © 2004, European Society of Cardiology

Overexpression of sarcolipin decreases myocyte contractility and calcium transient

Gopal J. Babu, Zhaolun Zheng, Poornima Natarajan, Debra Wheeler, Paul M. Janssen and Muthu Periasamy^*

Department of Physiology and Cell Biology, 304 Hamilton Hall, 1645 Neil Ave, The Ohio State University College of Medicine and Public Health, Columbus, OH 43210, United States

^* Corresponding author. Tel.: +1 614 292 2310; fax: +1 614 292 4888. Email address: periasamy.1{at}osu.edu

Received 25 March 2004; revised 18 August 2004; accepted 23 August 2004

	Abstract

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

 
Objective: Sarcolipin (SLN) is a novel 31-amino-acid protein associated with the sarcoplasmic reticulum (SR) whose function in cardiac muscle is poorly defined. In this study, we tested the hypothesis that SLN is a regulator of SR Ca²⁺ transport function by overexpressing SLN in adult rat ventricular myocytes which express low levels of SLN.

Methods: Expression of SLN mRNA in rat tissues was analyzed by Northern blot as well by RT-PCR analysis. To define the role of SLN in cardiac muscle contractility, we overexpressed SLN in adult rat ventricular myocytes using adenoviral gene transfer techniques. Localization of SLN in the adult rat ventricular myocytes was determined using confocal microscopy. Myocyte contractility and calcium transients were measured using edge detection and Fura 2AM.

Results: Our results demonstrate that overexpression of SLN decreased the cell shortening significantly when compared to control myocytes, whereas the time to peak contraction was not altered. In addition, SLN overexpression prolonged the time of 50% relaxation. Calcium transient analysis shows that time to 50% decay of [Ca²⁺]i was markedly prolonged in SLN-overexpressing myocytes (control –245.0 ± 3.78 vs. SLN –199.0 ± 3.25 ms, p<0.001). However, there were no significant differences in peak amplitudes of [Ca²⁺]_i between SLN-overexpressing and control myocytes. We further demonstrate that SLN is localized within the SR membrane similar to PLB and SR Ca²⁺ ATPase. Co-immunoprecipitation studies indicate that SLN can physically interact with phospholamban.

Conclusions: We conclude that SLN may play an important role in regulating the SR calcium ATPase pump, possibly by interacting with phospholamban.

KEYWORDS Sarcolipin; Calcium; SERCA; Myocyte; Adenovirus

	1. Introduction

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

 
Sarcolipin (SLN) is a low molecular weight protein that was originally identified to co-purify with the skeletal muscle sarcoplasmic reticulum calcium ATPase (SERCA) pump [1,2]. Recent studies showed that in human and rabbit, SLN mRNA is expressed both in heart and skeletal muscle but at higher levels in the skeletal muscles [3]. In smaller mammals such as mice, SLN mRNA is most abundant in atria and slow twitch muscle and is below detectable levels in the ventricle [4]. The exact role of SLN in cardiac physiology and pathology is not understood. A recent study showed that SLN mRNA was up-regulated 50-fold in the hypertrophied ventricles of Nkx2-5 null mice [5] suggesting that SLN may have important roles in the ventricle during certain pathophysiological conditions.

Although the exact role of SLN in cardiac calcium homeostasis and contractility is not yet fully defined, recent studies carried out in HEK cells showed that SLN can regulate SERCA pump activity [6–9]. Co-expression of SLN with either SERCA1a or SERCA2a decreases the apparent Ca²⁺ affinity of SERCA pump [3,7]. These studies suggest that SLN can inhibit SERCA function and lower the basal calcium stores in the sarcoplasmic reticulum. Structural similarity between SLN and phospholamban (PLB) indicates that they are homologous proteins with a potentially similar function [6,8–10]. Calcium uptake studies using microsomes isolated from HEK cells co-transfected with SERCA and SLN or PLB suggest that SLN is a less effective inhibitor than PLB. Furthermore, when SLN and PLB are co-expressed, SLN is shown to inhibit the polymerization of PLB resulting in more monomers of PLB and super inhibition of SERCA pump [7,9]. However, these studies were carried out in non-muscle cells (HEK 293 cell lines) which lack SR membrane structure. Recently, it was shown that cardiac-specific overexpression of SLN in mouse results in decreased contractile function and ventricular hypertrophy [11]. This study also raises the possibility that chronic overexpression of SLN can lead to pathophysiology and thus makes it difficult to understand its physiological relevance. Therefore, in this study we have used transient expression of SLN in ventricular myocytes by adenoviral gene transfer and studied its effect on calcium transient and myocyte contractility.

It is also important to emphasize that PLB, which regulates SERCA pump affinity for Ca²⁺ [12–14], is expressed much lower in atria than in ventricle [15,16]. This is in contrast to SLN expression which is higher in the atria compared to the ventricle [4]. Based on the expression pattern and structural similarity of these two proteins [6,8–10], we hypothesize that SLN and PLB may have similar functions. To better define the role of SLN in cardiac myocytes, we overexpressed SLN in adult rat ventricular myocytes (which normally express low levels of SLN) using adenoviral gene transfer techniques. We specifically addressed the following questions: (1) Does SLN localize in the SR membrane similar to SERCA and PLB? (2) How does the overexpression of SLN affect the myocyte contractility and calcium transients? (3) Does SLN physically interact with SERCA and/or PLB?

	2. Methods

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

 
All experiments were performed in accordance with the NIH guidelines and approved by the Institutional Laboratory Animal Care and Use Committee at the Ohio State University.

2.1. Messenger RNA analysis
Total RNA was isolated from various rat muscle tissues using the ULTRASPEC-II RNA Isolation System (Biotecx Laboratories, Houston, TX, USA). For the developmental study, pooled tissues of heart and skeletal muscle were used. RNAse protection analysis was carried out as described earlier [17] using radiolabelled RNA probes specific for rat SLN cDNA and L32, a ribosomal protein.

For Northern blot analysis, 10 µg of total RNA from rat atria, ventricle, fast-twitch (quadriceps) and slow-twitch (soleus) skeletal muscles was resolved on a 1% formaldehyde/agarose gel, transferred to nitrocellulose membrane, and hybridized with full-length rat SLN specific cDNA. The blot was stripped and probed for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA.

RT-PCR analysis was done using 5 µg of total RNA from various muscle tissues. Following oligo (dT) primed first-strand cDNA synthesis, a 2-µl portion of the first-strand cDNA mixture was subjected to PCR using primers specific for rat SLN cDNA (Forward 5'-GAGAAATTGAAGTCCACGGC-3' and Reverse 5'-GGTGTGACAGGCATTGTGAG-3') and GAPDH (Forward 5'-CCCATCACCATCTTCCAGGA-3' and Reverse 5'-TTGTCATACCAGGAAATGAGC-3').

2.2. Generation of NF-SLN adenovirus
The adenoviral construct containing either N-terminal FLAG tagged (NF)-SLN or SLN was generated using the method described by He et al. [18]. Rat sarcolipin cDNA was cloned in frame to FLAG tag and subcloned into the shuttle vector, pShuttle-CMV (Stratagene). Recombinant adenoviruses (Ad) containing NF-SLN or SLN were harvested 7–10 days later. The viral titers were determined by plaque assay with the final yield at 10¹⁰ to 10¹¹ pfu/ml. Adenovirus without transgene was used as a control.

2.3. Isolation of rat ventricular myocytes
Cardiac myocytes were isolated from adult Sprague Dawley rats as described previously [19]. The isolated cells were washed three times using phosphate buffered saline and plated in medium 199 (Gibco-BRL) supplemented with (in mmol/L) 5 creatine, 2 L-carnitine, 5 taurine and 0.2% BSA at a field density of 10,000 cells/cm² on 35-mm culture dishes precoated with laminin (Sigma). After 1 h, the media was changed to remove the nonadherent cells and then infected with control Ad.GFP (adenovirus without transgene) or recombinant adenovirus containing SLN or NF-SLN at a multiplicity of infection (MOI) of 50 for 48 h in serum-free DMEM.

2.4. Immunostaining of isolated cardiac myocytes
Isolated adult rat ventricular myocytes were infected with control or Ad.NF-SLN (multiplicity of infection 50) and processed for immunostaining and confocal imaging as described earlier [20]. Cells were incubated in primary antibody (rabbit polyclonal SERCA2a [17], mouse monoclonal FLAG antibody [Sigma]or -actinin antibodies [20]) in phosphate buffered saline (PBS) containing 2% normal goat serum and 1% Triton X-100 for 1.5 h. The glass coverslips were washed three times with PBS containing 1% Triton X-100. Cells were incubated with Texas Red-conjugated goat anti-rabbit and FITC-conjugated goat anti-mouse (Molecular Probes) secondary antibodies for 1 h followed by washing three times with PBS containing 1% Triton X-100. After immunostaining, cells were visualized by excitation at 488 nm for FITC and 543 nm for Texas Red using a Zeiss Laser Scanning Microscope (LSM510).

Indirect immunoflouresence for NF-SLN and PLB was done as previously described [21]. Briefly, myocytes on coverslips were fixed in 4% paraformaldehyde, washed with PBS, and treated with 50 mM ammonium chloride to remove the excess formaldehyde. Cells were blocked with 20% normal goat serum in 0.5% Triton X-100, and treated with anti-mouse PLB monoclonal antibody (Upstate) for 1.5 h. Cells were washed with PBS containing 0.5% Triton X-100 and blocked with 2% normal goat serum in 0.5% Triton X-100, and treated with secondary antibody conjugated to FITC (Molecular Probes) for 1 h. After washing, cells were incubated with 1:20 dilution of goat anti-mouse IgG antibody (Sigma) overnight at 4°C. Coverslips were then washed as above and incubated with a 1:20 dilution of goat anti-mouse Fab fragments (Jackson ImmunoResearch Laboratories) for 1.5 h. After blocking with 20% normal goat serum in 0.5% Triton X-100, cells were treated with anti-FLAG antibody (Sigma). Coverslips were washed, blocked, and incubated with a secondary antibody conjugated to Texas Red (Molecular Probes) as described above.

2.5. Myocyte shortening measurements
Cardiac myocyte shortening measurements were done as described earlier [22] using an IonOptix system. The myocytes were placed in a perfusion chamber on an inverted microscope stage in Krebs–Henseleit (K-H) solution containing (in mmol/L) 120 NaCl, 4.7 KCl, 0.94 MgSO₄, 1.2 KH₂PO₄, 25 NaHCO₃, 11.5 Glucose and 0.1 ascorbate, preoxygenated with 95% O₂/5% CO₂ with 1 mM Ca²⁺. Experiments were carried out at 37 °C with field stimulation at 1 Hz. Contraction and relaxations were monitored by a video edge-detection device. The output of the video edge detector was digitally recorded and analyzed offline. Myocytes used to determine the functional parameters were rod-shaped without spontaneous contraction and could react to the pacing and different reagents throughout the experiments. After equilibration in K-H solution containing 1 mM Ca²⁺ for 10 min, 10–15 myocytes were studied randomly as far as possible in each preparation and the contraction amplitude, velocities, time to peak contraction (TTP), and time to 50% relaxation (RT₅₀) of the paced myocytes were measured. The myocytes were then subjected to increased Ca²⁺ (2–4 mM) or isoproterenol (10^–10–10^–6 M) until a maximum contraction amplitude was reached (no increase in contraction amplitude after further increment) or arrhythmias were observed. Concentration response curves to isoproterenol were constructed in 1 mM Ca²⁺ with log unit increments from 0.1 nM. The contraction experiments for control myocytes were done whenever possible concurrently in the same preparation. Percentage shortening was defined as the changes in cell length divided by the resting cell length. Data were analyzed offline by Felix software (Photon Technology International) and custom programs written in Labview (National Instruments).

2.6. Calcium transient measurements
Calcium transients were measured using Fura-2AM as previously described [23]. Briefly, isolated myocytes were incubated with 5 µM Fura-2AM (Molecular Probes) and 0.05% pluronic F-127 (Molecular Probes) for 10 min and rinsed three times with K-H solution containing 1 mmol/L CaCl₂. The Fura-2AM loaded myocytes were kept at room temperature for 30 min before mounting. All experiments were performed at 37 °C and the myocytes were stimulated at 1 Hz as described above. Calcium transients in myocytes were monitored by collecting epifluorescence signals at 510-nm wavelength following dual excitation at 340 and 380 nm, and expressed as the ratio of the fluorescence intensities of these signals (R 340/380). Myocytes were chosen randomly and calcium transient data were analyzed with Felix software (Photon Technology International).

2.7. Western blot analysis
Total protein from Ad.NF-SLN-infected and control myocytes were isolated as described earlier [24]. NF-SLN levels were determined by Western blot using FLAG antibody (Sigma). To determine the levels of calcium handling proteins, total protein extracts from control and Ad.NF-SLN-infected myocytes were separated on SDS-PAGE and immunoblotted with antibodies specific for SERCA [17], PLB, calsequesterin, sodium–calcium exchanger (NCX), and dihydropyridine receptor 2 (DHPR2) [all antibodies were from Affinity BioReagents], as described earlier [15]. To determine the PLB pentamer/monomer ratio, unboiled protein samples were used for Western blot analysis.

2.8. Co-immunoprecipitation
After 48-h infection with Ad.NF-SLN, myocytes were suspended in a buffer containing (in mmol/L) 25 Tris, pH 7.4, 150 NaCl, 1 CaCl₂ and 1% Triton X-100 by gently pipetting and allowed to lyse on ice for 30 min. The cell lysate was centrifuged at 12,000 x g for 20 min at 4 °C and the supernatant was stored at –80 °C. Immunoprecipitation was carried out using Sigma FLAG Tagged protein immunoprecipitation kit (cat. #FLAGIPT-1). Briefly, about 1 mg of total protein isolated from Ad.NF-SLN myocytes was incubated on the Anti-FLAG M2 Affinity gel and washed extensively with Tris-buffered saline. The bound proteins were eluted with 0.1 M glycine pH 3.5 and analyzed by Western blot.

2.9. Statistics
Results were expressed as mean ± S.E. Statistical significance was estimated by paired or unpaired Student's t-test. One or two-way analysis of variance (ANOVA) was used for multiple comparisons when appropriate. A value of p<0.05 was considered statistically significant.

	3. Results

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

 
3.1. SLN expression is relatively abundant in the atrial chamber
We chose to determine SLN expression at the mRNA level because our attempts to generate a polyclonal antibody for SLN have not been successful. First, we determined the expression levels of SLN mRNA in rat species because we planned to use rat ventricular myocytes for the overexpression studies described below. Northern blot analyses (Fig. 1A) indicate that SLN is expressed at high levels in the atria and its expression was below detectable level in rat ventricle. In skeletal muscle tissues, SLN mRNA was expressed at low levels in soleus and was below detectable level in fast-twitch skeletal muscle. However, RT-PCR analysis (Fig. 1A) demonstrates that SLN mRNA is indeed expressed in the ventricle and fast-twitch skeletal muscles. We next analyzed the expression of SLN during cardiac and skeletal muscle development by RNAse protection analysis. Results show that the SLN mRNA appears around day 15 of postcoitum and increases in both cardiac and skeletal muscles. In the adult, its expression remains high in the atria and in soleus muscle (Fig. 1B).

View larger version (88K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Tissue distribution and developmental expression of SLN mRNA. (A) Northern blot analysis shows the high level expression of SLN transcript in rat atria. RT-PCR analysis shows the basal level expression of SLN transcript in ventricle and skeletal muscle tissues. (B) About 5 µg of total RNA isolated from embryonic, neonatal, and adult cardiac and skeletal muscles was hybridized with riboprobes specific for SLN and L32 (a ribosomal protein, as a control). The protected fragments were separated on a 5% denaturing PAGE and visualized following 24-h exposure. Signal intensities were determined by densitometry.

 
3.2. Adenoviral expression of SLN in ventricular myocytes did not alter the SERCA and PLB protein levels
For SLN expression, the adenoviral vectors carrying SLN coding sequence with or without N-terminal FLAG were infected at a multiplicity of infection (MOI) of 50 and the infection efficiency was assessed by the green fluorescent protein expression. The protein levels of NF-SLN were verified after 48 h of infection by Western blot analysis (Fig. 2A) using anti-mouse FLAG antibody. Since our attempts to generate an antibody against SLN were not successful, we could not measure the fold of NF-SLN expression in infected myocytes over endogenous SLN. For contractile measurements we used myocyte preparations demonstrating similar amounts of increased NF-SLN.

View larger version (48K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Western blot analysis showing the SERCA, PLB, and CSQ protein levels in control and Ad.SLN-infected myocytes. (A) Total protein isolated from myocytes after 48 h of Ad.SLN infection was boiled and resolved on SDS-PAGE and immunoblotted with antibodies specific for FLAG, SERCA2a, PLB, CSQ, DHPR2, NCX, and/or sarcomeric -actin. (B) Samples were unboiled and analyzed for PLB monomers (PLBM) and pentamers (PLBP). NS–nonspecific.

 
To determine if increased SLN expression modified the SERCA and PLB protein levels in Ad.NF-SLN-infected myocytes, quantitative Western blot analysis was carried out. Sarcomeric -actin was used as an internal loading control. Our results show that the expression levels of SERCA2a, PLB, and CSQ are similar in both uninfected and infected myocytes (Fig. 2A), indicating that expression of these proteins was not affected after 48 h of SLN overexpression. In addition, we observed that SLN overexpression did not alter the PLB pentamer/monomer ratio (Fig. 2B).

To determine whether SLN overexpression altered the expression of other calcium handling proteins, we quantitated the protein levels of NCX and DHPR2 by Western blot analysis. As seen in Fig. 2A, SLN overexpression did not affect the NCX and L-type calcium channel expression levels.

3.3. SLN and SERCA2a are co-localized in the cardiac SR membrane
To determine whether NF-SLN targets appropriately to the cardiac SR in transfected myocytes, we performed immunostaining and confocal microscopy using FLAG antibody. Control myocytes showed no staining with FLAG antibody (Data not shown), whereas Ad.NF-SLN-infected myocytes showed a distinctive horizontal and vertical pattern (Fig. 3, Panel A, green and Panel E, red), which was indistinguishable from that seen with SERCA2a (Panel B, red) and PLB (Panel D, green) antibody staining. Our data thus demonstrate that SLN is localized within SR membrane and its distribution pattern is similar to SERCA2a and PLB (Fig. 3, yellow color in Panel C and Panel F) indicating that it may be co-localized with SERCA and PLB.

View larger version (70K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Confocal microscopic images of rat ventricular myocytes showing the co-localization of SLN, SERCA2a, and PLB. Ventricular myocytes were infected with Ad.SLN and stained with FLAG antibody (A and E) and SERCA2a antibody (B) or PLB antibody (D). Panel C–overlay of images A and B; panel F–overlay of images D and E.

 
3.4. Adenoviral expression of SLN decreases the cardiac myocyte contractility
Next we determined the effect of SLN overexpression on cardiac myocyte contractility. Ad.NF-SLN or control adenovirus infected ventricular myocytes were loaded with Fura 2AM and paced at 1 Hz and simultaneously cell shortening and calcium transients were measured. The number of myocytes with visible contractions in response to 1 mM [Ca²⁺]_o was compared between Ad.NF-SLN.GFP-infected and Ad.GFP-infected (control) groups. At this Ca²⁺ concentration, more than 80% of myocytes in both groups contracted.

The cell shortening was 30.9% lower in myocytes infected with Ad.NF-SLN (5.14 ± 0.14% in NF-SLN group, and 7.44 ± 0.21% in control group, p<0.001) whereas the time to peak contraction was not significantly different (107.4 ± 1.92 ms NF-SLN group, and 111.1 ± 1.72 ms in control group, p>0.05) (Fig. 4A and B). In addition, Ad.NF-SLN-infected myocytes exhibit a prolonged time to 50% relaxation (86.87 ± 1.83 ms in NF-SLN group, and 71.35 ± 1.54 ms in control group, p<0.001) (Fig. 4C). These results suggest that overexpression of SLN decreased the rates of relaxation. These experiments were performed using both Ad.SLN with or without FLAG tag and the results were very similar (data not shown), indicating that the addition of N-terminal FLAG tag did not affect SLN function.

View larger version (14K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 SLN overexpression decreases the myocyte contractility. Contraction amplitude (A), time to peak contraction (B) and time to 50% relaxation (C) in myocytes from control and SLN-overexpressing adult rat ventricular myocytes at 1 mM CaCl2. Myocytes were studied 48 h after Ad.SLN infection. **Significantly different from control myocytes (p<0.001), n=30.

 
3.5. Adenoviral expression of SLN alters calcium transient and decay
To determine how SLN overexpression modifies Ca²⁺ handling, we measured the peak value, half-time of relaxation (t_1/2), and time to peak-value of calcium transient. Our results show that overexpression of SLN resulted in a slowed Ca²⁺ transient decay (Fig. 5A). The time to 50% decay of calcium was markedly prolonged in SLN-overexpressing myocytes compared with control (Fig. 5B; 245.0 ± 3.78 vs. 199.0 ± 3.25 ms, p<0.001), indicating slower SR Ca²⁺ reuptake. However, time to peak (TTPc) was unchanged between SLN-overexpressing and control myocytes which is consistent with the myocytes shortening parameters (Fig. 4B). Surprisingly, there were no significant differences in calcium transient amplitudes between Ad.NF-SLN-infected and control myocytes (1.665 ± 0.025 vs. 1.622 ± 0.025, p>0.05). This may be due to the slow kinetics of the intracellular Ca²⁺ indicator Fura 2AM or that differences in SR calcium load between these two groups are not significant enough for detection by the indicator.

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 SLN overexpression alters calcium transient. (A) Sample trace of calcium transients recorded in control and Ad.SLN-infected myocytes. (B) Time to 50% decay of calcium was significantly prolonged in SLN-infected myocytes. Calcium transients were recorded at 37 °C. **Significantly different from control myocytes (p<0.001) n=30.

 
3.6. Adenoviral expression of SLN does not modify myocyte response to Ca²⁺ and isoproterenol
We wanted to test whether SLN is involved in the β-adrenergic receptor-mediated effect on myocyte contractility. To test this idea, Ad.NF-SLN-infected and control myocytes were treated sequentially with isoproterenol (10^–10 to 10^–6 mM) and the contractile function was measured. The contraction amplitude was not significantly different between control and SLN-overexpressing myocytes at the maximally effective concentration of isoproterenol (Fig. 6A). In addition, we compared the contraction amplitudes of Ad.NF-SLN-infected myocytes with control myocytes at different concentrations of Ca²⁺. The contraction amplitude was decreased in SLN-overexpressing myocytes and the differences were more pronounced at higher Ca²⁺ concentrations (1–4 mM, Fig. 6B).

View larger version (14K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Effect of isoproterenol and calcium on myocyte contractility. Concentration response curve to isoproterenol was constructed in 1 mM Ca2+ as described in Methods. The contraction amplitude was not significantly different between control and SLN-overexpressing myocytes in maximally activating isoproterenol (10–10 to 10–6 M) [Panel A] whereas it is decreased at high calcium (1–4 mM) in SLN-overexpressing myocytes [Panel B]. n=25.

 
3.7. SLN co-immunoprecipitates PLB
To test the hypothesis that SLN interacts with PLB and SERCA, we performed co-immunoprecipitation analysis using FLAG antibody. Results shown in Fig. 7 demonstrate that FLAG antibody against NF-SLN co-immunoprecipitates PLB. However, we did not find SERCA2a in the co-immunoprecipitate (data not shown). These results are consistent with the previous reports showing that PLB and NF-SLN form a binary complex and inhibit SERCA function [9,25].

View larger version (72K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 SLN co-immunoprecipitates PLB. Total protein isolated from Ad.SLN-infected myocytes was immunoprecipitated with FLAG antibody and analyzed by Western blot analysis using PLB antibody. Lane 1–total protein from ventricular myocytes; lane 2–final wash before elution; lanes 3 and 4–FLAG antibody immunoprecipitates. PLBP–PLB pentamer and PLBM–PLB monomer.

 

	4. Discussion

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

 
In the present study, we demonstrate that adenoviral overexpression of SLN in rat ventricular myocytes decreases the myocyte contractility and calcium transients. We also present evidence that SLN is localized in the cardiac SR membrane and shows a distribution pattern similar to SERCA2a. Further, we show that SLN can physically interact with phospholamban. These data provide important evidence that SLN may play a critical role in regulating the cardiac SR calcium transport function possibly by interacting with phospholamban.

SLN expression has been studied in many different species. In human and rabbit, SLN mRNA is expressed at higher levels in the skeletal muscles than in the heart [3], whereas in mice SLN mRNA is most abundant in atria and slow twitch muscle and is below detectable levels in the ventricle [4]. Our data show that in the adult rat heart SLN mRNA is expressed at high levels in the atria but below detectable levels in the ventricle. This observation contradicts an earlier study which reports higher levels of SLN in the ventricle using RT–PCR [26]. Our observations and recent studies in mouse, rabbit, and humans [3,4] suggest that SLN expression is species-specific and differentially regulated in skeletal and cardiac muscles in smaller vs. larger mammals. In addition, a recent study showed that SLN mRNA was up-regulated 50-fold in the hypertrophied ventricles of Nkx2-5 null mice [5]. This finding, however, needs further validation in other models of heart disease. These results taken together suggest that SLN may play an important role in regulating myocyte function both in the atria and ventricle and as well during cardiac hypertrophy, a condition in which both calcium transport and contractile function are altered.

The major findings of our study demonstrate that overexpression of SLN prolonged the time to 50% decay of calcium indicating a slower calcium reuptake by the SR. These results are consistent with the idea that SLN inhibits SERCA2a, which leads to decreased rates of calcium uptake and slowed muscle relaxation. Our results are further supported by recent studies carried out by MacLennan and co-workers [6,7,25]. Overexpression of NF-SLN in mouse skeletal muscle resulted in significant reductions in both twitch and tetanic-peak force amplitude and maximal rates of contraction and relaxation and increased fatigability with repeated electrical stimulation [27]. In addition, Asahi et al. [11] recently reported that overexpression of SLN in the mouse heart led to decreased contractility and ventricular hypertrophy. SLN overexpression led to a decrease in peak amplitude of calcium transients in TG papillary muscle. These studies collectively suggest that impaired myocyte contractility could be due to the inhibition of SERCA function. Our results further demonstrate that SLN overexpression does not induce changes in the expression of major calcium handling proteins including SERCA, PLB, NCX, and L-type calcium channel. However, the effect of SLN overexpression on calcium sensitivity of thin-filament proteins has not been ruled out.

The detailed mechanism of SLN action on SR calcium transport remains to be elucidated including its dynamic interaction with PLB and SERCA pump. In this study, we demonstrate for the first time that SLN is localized in the SR membrane similar to SERCA2a and PLB. These results indicate that SLN, SERCA2a, and PLB may co-localize in cardiac myocytes. In a recent study Asahi et al. [7,25] reported that SLN can affect PLB pentamer levels, generating more monomers, an inhibitory form [28,29]. Although SLN interacts with PLB pentamer, we did not find any alteration in the PLB pentamer/monomer ratio in SLN-overexpressing myocytes. Because a number of conditions may affect PLB pentamer/monomer ratio including sample preparation and electrophoresis, these findings may require additional experimentation. The exact mechanism of SLN and PLB interaction and how they might affect SERCA function is of significant interest. Our future studies will address if SLN can disrupt the PLB pentamer and/or interact directly with SERCA pump.

There is discordance between PLB and SLN expression within the heart: SLN is high in the atria (Fig. 1) [4] where PLB level is low [15,16]. In contrast, SLN is below detectable levels in the ventricle where PLB is very high. In addition, SLN is expressed at high levels in fast-twitch skeletal muscle [3], a tissue which does not express phospholamban. Therefore, it is also possible that SLN action does not depend on PLB. This would suggest that SLN may directly interact with SERCA pump in the absence of PLB. Future studies will be directed towards understanding the mechanism of SLN interaction with SERCA pump and its role in SR calcium homeostasis.

	Acknowledgement

 
This work was supported by National Institutes of Health Grant HL-64140 (to M.P.) and American Heart Association Grant (to G.J.B.).

	Notes

 
Time for primary review 19 days

	References

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

 

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