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J. Biol. Chem., Vol. 281, Issue 7, 3972-3979, February 17, 2006
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1
From the
Department of Physiology and Cell Biology, The Ohio State University College of Medicine and Public Health, Columbus, Ohio 43210 and the Departments of Surgery and ¶Molecular Genetics and Biochemistry, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267
Received for publication, September 7, 2005 , and in revised form, December 19, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-adrenergic agonists. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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SLN is a 31-amino acid protein expressed in both cardiac and skeletal muscle (11, 13–15). We have recently demonstrated that SLN is localized in the cardiac SR membrane, and its distribution pattern is similar to SERCA2a and PLB (11). SLN mRNA is differentially expressed in small as opposed to larger mammals. In rodents, SLN mRNA is abundant in the atria with very low levels in the ventricle and skeletal muscles (11, 14, 15). In contrast, in larger mammals including humans, SLN mRNA is abundant in fast-twitch skeletal muscle compared with atria and ventricle (13). SLN expression is developmentally regulated (11), and its expression levels are modified under certain pathological conditions of the muscle (16, 17). Decreased expression of SLN mRNA has been shown in the atria of patients with atrial fibrillation (16). A recent study also showed that SLN mRNA was up-regulated 
50-fold in the hypertrophied ventricles of Nkx2–5-null mice (17).
Structural similarities between SLN and PLB indicate that they are homologous proteins and may functionally substitute for each other (9, 10, 18, 19). Recent studies carried out in HEK cells showed that SLN could inhibit the SERCA pump activity (8, 18). Co-expression of SLN with either SERCA1a or SERCA2a decreases the apparent Ca2+ affinity of the SERCA pump. Furthermore, when SLN and PLB are co-expressed, SLN was shown to inhibit the polymerization of PLB, resulting in more monomers and super-inhibition of the SERCA pump (8, 20). Using adenoviral gene transfer into cardiac myocytes, we recently demonstrated that overexpression of SLN resulted in decreased myocyte contractility and calcium handling. However, overexpression of SLN did not alter the PLB pentamer/monomer ratio in cardiac myocytes (11).
Based on our adenoviral gene transfer studies in cardiac myocytes (11), we hypothesized that SLN can directly modulate SERCA pump activity and affect cardiac contractility. To test this hypothesis, we specifically altered the SLN to SERCA ratio in the ventricle by overexpressing SLN using the 
-MHC gene promoter. Results presented in this study suggest that SLN directly inhibits the SERCA pump activity, and its inhibitory effect can be reversed upon adrenergic stimulation and increased frequency.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Generation of Transgenic Mice—N-terminal FLAG-tagged mouse SLN cDNA (11) was amplified by PCR and ligated into the SalI and HindIII sites downstream of the 5.5-kb mouse -MHC promoter and upstream of the poly(A) signal sequence from the human growth hormone. The complete recombinant construct was excised from the plasmid backbone by NotI restriction digestion and gel-purified. To generate transgenic founder mice, DNA samples were microinjected into the pronuclei of C57BL/6 murine embryos at the core facility for transgenics, University of Cincinnati.
Mice carrying the transgene were identified by PCR analysis using primers specific for -MHC (5'-GCCCACACCAGAAATGACAGA-3') and the antisense primer specific for the 3'-end of SLN cDNA (5'-TCAGTATTGGTAGGACCTCA-3'). The copy number of the transgene was identified by Southern blot analysis of DNA samples from TG mice as described earlier (23).
Determination of SERCA/SLN Ratio by RT-PCR—Total RNA was isolated from ventricle or atria (pooled from two mice) using the ULTRASPEC-II RNA Isolation System (Biotecx Labs., Houston, TX). RT-PCR analysis was done using 1 µg of total RNA from ventricle or atria as described earlier (22). Following oligo(dT)-primed first-strand cDNA synthesis, 1-µl portions of the first-strand cDNA mixture were subjected to PCR using primers specific for mouse SERCA (forward, 5'-CTGTGGAGACCCTTGGTTGT-3' and reverse, 5'-CAGAGCACAGATGGTGGCTA-3'), mouse SLN (forward, 5'-GCACTAGGTCCTTGGCATGT-3' and reverse, 5'-ACTCAAGGGACTGGCAGAGA-3'), NF-SLN (FLAG forward, 5'-CTACAAGGACGACGATGACAA and human growth hormone poly(A) reverse, 5'-AGGTTGTCTTCCCAACTTGC) and mouse GAPDH (forward, 5'-CCCATCACCATCTTCC AGGA-3' and reverse, 5'-TTGTCATACCAGG AAATGAGC-3'). PCR was adjusted to obtain equal amounts of SERCA2a, and the number of cycles was chosen to fall within the exponential phase of amplification. Total SLN mRNA levels were calculated by adding endogenous and NF-SLN mRNA levels. The PCR protocols were as follows: 94 °C for 30 s, 55 °C for 30 s, and 75 °C for 60 s (35 cycles) with a 72 °C extension for 7 min.
Western Blot Analysis—Cardiac homogenate was prepared from transgenic and non-transgenic ventricles, and Western blot analysis was carried out as described earlier (11, 23). Briefly, equal amounts of total homogenates from SLN TG and NTG ventricles were separated on: 5% (for RyR, NCX, and PMCA) 8% (for SERCA and CSQ), 10% (for DHPR2 and triadin), and 14% (for PLB and NF-SLN) SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were immunoprobed with the following primary antibodies: anti-rabbit SERCA2a, anti-rabbit PLB, anti-rabbit CSQ (ABR), anti-mouse DHPR2, anti-rabbit triadin, anti-mouse PMCA (ABR), anti-mouse NCX (Swant, Switzerland), anti-rabbit S16 or T17 PLB antibody (Cyclacel, Dundee, UK). Protein loading was normalized to Coomassie Blue staining and -actin levels. Signals were detected by SuperSignal WestDura substrate (Pierce) and quantitated by densitometry.
To determine -adrenergic agonist-mediated PLB phosphorylation, SLN TG hearts were perfused with isoproterenol in an isolated work-performing heart setup as described below. One set of hearts was freeze-clamped after 30 min without isoproterenol, and the other set was treated with isoproterenol (1 µM) for 5 min after 25 min of perfusion. PLB phosphorylation was estimated by Western blotting analyses.
Calcium Uptake Assay—Ventricles from TG and NTG mice were used for calcium uptake assays as described earlier (23, 24). Briefly, ventricular tissue was homogenized in 8 volumes of protein extraction buffer (in mmol/liter, 50 KPi, 10 NaF, 1 EDTA, 300 sucrose, 0.5 dithiothreitol, and 0.3 phenylmethylsulfonyl fluoride), and calcium uptake was measured by the Millipore filtration technique. Ventricular homogenates (150 µg) from NTG and SLN TG animals were incubated at 37 °C in a 1.5 ml of calcium uptake medium (in mmol/liter, 40 imidazole, pH 7.0, 100 KCl, 5 MgCl2, 5NaN3, 5 potassium oxalate, and 0.5 EGTA) and various concentrations of CaCl2 to yield 0.03–3 µmol/liter free Ca2+ (containing 1 µCi/µmol 45Ca2+). To obtain the maximal stimulation of SR Ca2+ uptake, 1 µM ruthenium red was added immediately prior to the addition of the substrates to begin the calcium uptake. The reaction was initiated by the addition of 5 mM ATP and terminated at 1 min by filtration. The rate of calcium uptake and the Ca2+ concentration required for half-maximal velocity of Ca2+ uptake (EC50) were determined by non-linear curve fitting analysis using GraphPad PRISM 4.0 software.
Isolated Work-performing Heart Preparations—Work-performing heart preparations were performed as described previously (23, 25). Mice were anesthetized via intraperitoneal injection with 100 mg/kg sodium nembutal and 1.5 units of heparin to prevent intracoronary microthrombi. The aorta was cannulated, and retrograde perfusion (Langendorff mode) was carried out at a constant perfusion pressure of 50 mmHg with Krebs-Henseleit buffer containing (in mmol/liter) 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 0.5 Na-EDTA, 25 NaHCO3, 1.2 KH2PO4, and 11 glucose. All perfusion buffers were equilibrated with 95% O2 plus 5% CO2, yielding a pH of 7.4. A water-filled catheter (P-50) was inserted through mitral valve into the left ventricle. After a short period of stabilization on retrograde perfusion, the pulmonary vein was cannulated, and perfusion of the heart was switched from retrograde to anterograde. A 20-gauge cannula was tied into the left pulmonary vein to accommodate regulation on recording of venous return. Anterograde work-performing perfusion was initiated at a workload of 250 mmHg ml/min, which was achieved using a custom micrometer-controlled venous return of 5 ml/min and an aortic pressure of 50 mmHg. After establishment of the base line, responses to infusion of -adrenergic receptor agonist, isoproterenol, by a microperfusion pump (Master flex) were measured with varying concentrations for 2 min. The signals were digitized, and the following indices of cardiac performance were measured off-line using Biobench software (National Instruments, Inc): left ventricular systolic pressure, end diastolic pressure, diastolic pressure, the minimum (–dP/dt) and maximum (+dP/dt) derivatives of left ventricular pressure, time to peak systolic pressure (TPP), and time to reach 50% of relaxation (TR1/2). TPP and TR1/2 were normalized with respect to peak left ventricular pressure because they are dependent upon extent of pressure development. Force-frequency relationship was carried out to assess frequency-dependent contractile reserve. For these experiments, hearts were paced with frequencies from 4 to 12 Hz, and +dP/dt and –dP/dt were determined at multiple intervals.
Preparation of Muscle Fibers and Experimental Set-up—Small, unbranched trabeculae or the smallest of the RV papillary muscles were dissected from the right ventricle as previously described (26). The dimensions of the preparations were 221 ± 21 µm, 174 ± 18, and 1401 ± 78 (width x thickness x length in µm, n = 15) and are not different between the two groups. Using the dissection microscope, muscles were mounted between a platinum-iridium basket-shaped extension of a force transducer (KG7, Scientific Instruments GmbH, Heidelberg, Germany) and a hook (valve end) connected to a micromanipulator. Muscles were superfused with Krebs-Henseleit buffer ([Ca2+]o 1.5 mmol/liter) at 37 °C and stimulated at baseline (4 Hz). Muscles were stretched to a length where a small increase in length resulted in nearly equal increases in resting tension and active developed tension (26). This length was selected to be comparable to a length close to the end of diastole.
After stabilization, contractile parameters were recorded at 4 different muscle lengths between slack and optimal length, stimulated at rates between 4 and 14 Hz in a second protocol, and finally the response to a concentration-response curve of isoproterenol was assessed. SR calcium load was estimated via rapid cooling contracture (RCC) experiments (27).
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Simultaneous Measurement of Ca2+ Transient and Myocyte Shortening—Ventricular myocytes from SLN TG and NTG hearts were isolated as described previously (28). Briefly, mice were injected intraperitoneally (IP) with 0.04 ml of heparin (10,000 units/ml) 20 min before a 0.2-ml IP injection of sodium pentobarbital until unreactive. After aortic cannulation, heart was mounted on Langendorff apparatus and cleared of all blood with modified MEM (Sigma M0518, 37 °C, bubbled with 95%O2, 5%CO2). The heart was then perfused with blenzyme solution (Roche Applied Science, 37 °C, bubbled with 95%O2, 5%CO2). The tissue was then dissected in a Petri dish with fresh blenzyme solution, removing atria, and chopping into fine pieces. After triturating with a large bore pipette, the remaining pieces were post-digested with additional blenzyme solution and filtered through a 200 micron nylon mesh. All myocyte suspensions were spun for 20 s at 1000 rpm, supernatant decanted, and resuspended in modified MEM to which 0.1% bovine serum albumin and 50 µM Ca2+ were added. After gravity-settled, cells were resuspended in MEM containing 200 µM Ca2+.
Myocytes were loaded at 22 °C with Fluo-4 AM (10 µM, Molecular Probes, Eugene, OR) for 30 min for intracellular de-esterification. The instrumentation used for cell fluorescence measurement was a Cairn Research Limited (Faversham, UK) epifluorescence system. Myocytes were field-stimulated and superfused with (in mM/liter): 140 NaCl, 4 KCl, 1 MgCl2, 1 CaCl2, 10 glucose, and 5 HEPES (pH 7.4). [Ca2+]i was measured by Fluo-4 epifluorescence with excitation at 480 ± 20 nm and emission at 535 ± 25 nm. The illumination field was restricted to a small spot to get emission from a single cell. Data were expressed as F/Fo, where F is the fluorescence intensity and Fo is the intensity at rest. Simultaneous measurement of shortening was also performed using an edge detection system (Crescent Electronics, Sandy, UT). Data were expressed as % of resting cell length. Myocytes were field-stimulated via platinum electrodes connected to a Grass S48 stimulator, with Ca2+ transients and myocyte shortening simultaneously measured (29). Force-frequency relationship (0.2, 0.5, 1, and 2 Hz) and isoproterenol dose-response curves were generated.
Statistics—Results are expressed as mean ± S.E. Statistical significance was estimated by a paired Student's t test. A value of p < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-MHC promoter (Fig. 1A). The TG mice were generated in a CB56 background and the progeny screened for germline transmission of the transgene. PCR analysis indicated that 4 of 52 of initial F0 mice carried the transgene. Out of 4 founders, only 2 of them (line 20 and line 26) were fertile and produced progeny. Southern blot analysis revealed mouse lines 20 and 26 carried 1 and 2 copies of the transgene, respectively (data not shown). Transgenic mice were born in the expected Mendelian ratio and were indistinguishable from their NTG control littermates; there were no signs of phenotypic alterations or reduced viability.
Western blot analysis of NF-SLN expression in various muscle tissues including cardiac, skeletal, and vascular smooth muscle indicates that NF-SLN expression is restricted to heart and not detectable in other tissues analyzed (Fig. 1B). Further our data suggest that NF-SLN expression is 3-fold higher in the ventricle than in the atria of TG mice. This finding may suggest that high levels of endogenous SLN present in the atria may limit the amount of transgenic NF-SLN expression.
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2-fold higher NF-SLN protein than line 20. The transgenic mouse line 26 at 18–25 weeks old was chosen for further studies. To determine whether TG expression of NF-SLN has modified the levels of endogenous SLN, RT-PCR analysis was carried out, because we did not have a suitable antibody for SLN at that time. Our results show that overexpression of NF-SLN did not alter the endogenous SLN mRNA levels in both atria and ventricle (data not shown). We additionally wanted to determine the relative ratio of SLN to SERCA2a in the NTG and TG atria and ventricle. The data shown in Fig. 1D indicate the percent expression level of SLN mRNA to SERCA2a mRNA levels. Our results suggest that the ratio of SLN/SERCA is higher in atria than in the ventricle. In the transgenic ventricle, the ratio of SLN to SERCA was increased 57% more (from 107.35 ± 2.3% to 169.47 ± 2.7%) indicating that we altered the SLN to SERCA2a ratio successfully.
Overexpression of SLN Is Not Associated with Any Cardiac Pathology—We next examined whether overexpression of SLN led to any structural abnormalities and caused muscle pathology. There was no difference in the heart weight to body weight ratio in SLN TG mice when compared with age- and sex-matched littermate controls. Histological analysis of hearts from lines 20 and 26 at the age of 3, 6, and 12 months (via conventional microscopic evaluation of hematoxylin/eosin and Masson's trichromestained slices) revealed no difference in tissue morphology or evidence of fibrosis (Data not shown).
Expression Levels of SR Calcium-handling Proteins Are Unchanged in Transgenic Mice—Quantitative Western blot analysis was carried out to determine if increased SLN expression affected the levels of SERCA and PLB in the TG ventricle. Our results show that the expression levels of SERCA2a, PLB, and CSQ were unchanged in the TG ventricle (Fig. 2A) indicating that expression of these proteins was not affected by SLN overexpression. We also quantitated the expression levels of ryanodine receptor (RyR), L-type calcium channel subunit-dihydropyridine receptor 2 (DHPR2), and triadin to determine changes in Ca2+ release and entry mechanisms. As shown in Fig. 2B, SLN overexpression did not affect the RyR, DHPR2, or triadin levels. To determine whether SLN inhibition of the SERCA pump is associated with compensatory changes in the expression of other plasma membrane calcium extrusion systems, we quantitated the sodium-calcium exchanger (NCX) and plasma membrane calcium ATPase (PMCA) levels. Results in Fig. 2B indicate that SLN overexpression did not alter the expression of NCX and PMCA protein levels.
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SLN Overexpression Decreases the Apparent Ca2+ Affinity and Rate of Ca2+ Uptake—To determine the effect of the increased SLN to SERCA ratio on SR calcium transport, the rate of calcium dependence of calcium uptake was measured in total ventricular homogenates from SLN TG and NTG ventricles. Results show that there is a significant rightward shift in the sigmoid curve measuring calcium dependence of calcium uptake in TG mice indicating a reduced Ca2+ affinity in SLN TG ventricles (Fig. 4). The EC50 value for Ca2+ increased significantly in the TG ventricle (NTG, 163.8 ± 12.64 nM versus SLN TG, 209.3 ± 20.62 nM; n = 4; p < 0.05) when compared with the NTG ventricle. However, the maximum velocity (Vmax) of Ca2+ uptake was not significantly different between NTG and TG ventricles (NTG, 84.92 ± 13.86 nM versus SLN TG, 74.24 ± 13.95 nM n = 4; p < NS).
SLN TG Hearts Showed a Decreased Cardiac Performance in Isolated Work-performing Heart Preparations—The functional consequences of SLN overexpression in the heart were determined by measuring indices of cardiac performance with the anterograde-perfused work-performing heart preparations. The SLN TG hearts showed significant decreases in the maximum rate of contraction (+dP/dt) and relaxation (–dP/dt) compared with NTG hearts (Table 1). A tendency toward decreased baseline systolic and diastolic pressure was also observed in TG hearts; however, these decreases were not statistically different from NTG hearts. The other parameters of cardiac function such as time to peak pressure and half-relaxation pressure derived from intraventricular pressure tracings were not altered (Table 1).
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Contractility Is Restored in Isoproterenol-stimulated TG Hearts—It was of significant interest to determine how an increase in SLN levels affect the hearts ability to respond to -adrenergic stimulation. Both NTG and TG hearts responded to increasing doses of isoproterenol with increase in contractility as well as relaxation parameters (Fig. 5B). Interestingly, diminished base line +dP/dt and –dP/dt were restored to the level of control hearts after infusion of high doses of isoproterenol.
The phosphorylation status of PLB at Ser16 and Thr17 was estimated using quantitative Western blot analysis and phosphospecific PLB antibodies in isoproterenol-treated hearts as described under "Experimental Procedures." Our results showed that PLB phosphorylation was increased significantly in response to isoproterenol, but there was no difference between NTG and TG hearts (Fig. 5C).
Contractility Is Decreased in Muscle Preparations from SLN TG Hearts—At a base frequency of 4 Hz, length-dependent activation was similar between muscles from SLN TG and NTG littermates. Upon an increase in muscle length, active developed force increased in parallel. At optimal lengths, the developed force was lower, but not significantly, in TG versus NTG mice. Force-frequency (FF) behavior in NTG mice was biphasic. At lower rates of stimulation, a small positive FF relationship was observed, after which force declined upon further increase in stimulation frequency. In SLN TG mice, the loss of developed force with increasing frequency was larger, resulting in lower force development compared with NTG mice at 12 and 14 Hz. From Fig. 6A, it can be seen that at 4 Hz, the speed of contraction was slower in TG mice at low frequency, but not different at 14 Hz.
To determine the effect of isoproterenol on muscle contraction, a typical isoproterenol dose-response behavior was recorded. Under maximal and near-maximal concentration, force development was impaired in TG muscle compared with NTG. However, at maximal isoproterenol stimulation, the differences noted for the speed of contraction and relaxation, observed at 4 Hz between the groups became insignificant (Fig. 6B).
In addition, RCC experiments were carried out to determine the SR Ca2+ load. At a baseline frequency of 4 Hz, TG mice showed an RCC amplitude of 7.29 ± 1.76 mN/mm2 compared with 11.65 ± 1.62 mN/mm2 in NTG mice (NS, p = 0.12). At a stimulation of 12 Hz, TG mice showed a decreased RCC amplitude of 6.13 ± 1.56 mN/mm2 (n = 6), compared with the NTG littermates (11.03 ± 1.19 mN/mm2, n = 7, p < 0.05). These data suggest that overexpression of SLN decreases SR calcium load.
Overexpression of SLN Decreases Ca2+ Transient Amplitude and Slows Relaxation in Ventricular Myocytes—The effect of SLN overexpression on myocyte Ca2+ handling was studied using isolated ventricular myocytes from TG and NTG hearts (Fig. 7). Myocytes from SLN TG ventricles showed a decrease in Ca2+ transient amplitude by 57% (Fig. 7B) and significantly prolonged the rate of relaxation (NTG, 163 ± 14 ms versus TG, 255 ± 16 ms). In addition, shortening data paralleled the Ca2+ transient data (data not shown). Interestingly, -adrenergic stimulation with isoproterenol restored calcium transient amplitude in TG myocytes to the same level as NTG control myocytes (Fig. 7, A and B). Myocyte relaxation was also similar after isoproterenol stimulation (Fig. 7C). We further investigated the force-frequency relationship in myocytes isolated from SLN TG and NTG ventricle. At lower frequencies, there was a significant difference in Ca2+ transient amplitude and relaxation (0.2 Hz, NTG: 429 ± 31 ms versus SLN TG, 520 ± 43 ms, p < 0.05). However, at higher frequencies, the relaxation times were similar in both groups (2 Hz, NTG: 170 ± 7ms versus SLN TG, 171 ± 4ms, p = NS). In addition, Ca2+ transient amplitude and shortening at lower frequencies was significantly reduced in TG versus NTG myocytes, but similar at higher frequencies (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-adrenergic agonist, isoproterenol, suggesting that SLN acts as a reversible inhibitor similar to PLB.
Our studies additionally confirm many observations reported by Asahi et al. (12), in which rabbit SLN was expressed in the mouse by targeting a single copy of the -MHC-NF-SLN construct into the Hprt locus of the x chromosome. Targeting of the SLN into the HPRT locus resulted in heterogeneous SLN expression in female mice because of x-inactivation. This study showed that overexpression of rabbit SLN in mouse hearts reduced the apparent Ca2+ affinity of the SERCA pump. In vivo measurements of cardiac function showed significant decreases in +dP/dt and –dP/dt and leads to ventricular hypertrophy. They concluded that SLN inhibits the SERCA pump by stabilizing SERCA2a-PLB interaction and inhibiting PLB phosphorylation. This inhibition could be reversed upon -adrenergic agonist-mediated PLB phosphorylation. These studies taken together allowed us to reach similar conclusions that SLN overexpression leads to decreased SERCA2a pump affinity and contractile function of the heart. In contrast, the overexpression of mouse SLN in the mouse ventricle described here did not induce cardiac hypertrophy/heart failure. The development of cardiac hypertrophy observed by Asahi et al. (12) may very well be caused by the overexpression of rabbit SLN in the mouse heart, which differs from the mouse, at the N-terminal region (13).
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-adrenergic stimulation. However, in the present study, we did not see an appreciable difference in the -adrenergic agonist-mediated phosphorylation of PLB between the NTG and TG ventricle. These results suggest that SLN could play a direct role in mediating the -adrenergic response in the SLN TG hearts. SLN has a conserved threonine (Thr5) residue at the N terminus that can be phosphorylated during -adrenergic stimulation by serine/threonine kinases such as CaMKII, which may relieve its inhibitory effect on SERCA pump. Mutation of Thr5 to Ala leads to a slight gain in inhibitory function (18) further supporting the idea that phosphorylation of Thr5 could play a role in regulating SLN function. Recent studies also suggest that there are additional mechanisms independent of PLB phosphorylation, which may play a significant role in mediating the positive inotropic effects of the -adrenergic agonist (30) and force-frequency-dependent relaxation (31). However, the potential role of Thr5 as a target for calcium/calmodulin-dependent protein kinase II or protein kinase A phosphorylation during -adrenergic stimulation and increasing frequency needs to be demonstrated.
MacLennan and co-workers (9, 20) have demonstrated that SLN can form a binary complex with PLB. In a recent study we have also observed that PLB can be co-immunoprecipitated with NF-SLN (11). These studies suggest a physical interaction between SLN and PLB, and such a binary complex may enhance its inhibitory effect on SERCA (9). Phosphorylation of PLB during -adrenergic stimulation could dissociate the PLB-SLN binary complex from SERCA2a as effectively as it would remove PLB alone. It has also been suggested that SLN interaction with PLB could prevent PLB polymerization resulting in an increase in the active form, the monomer (8) thus promoting super-inhibition of SERCA pump. However, this model does not apply to situations where PLB is very low (atria) or non-existent such as in fast-twitch skeletal muscle. Further, our published data (11) and results from this study show that overexpression of SLN did not alter PLB levels, monomer to pentamer ratio, or its phosphorylation status. Therefore it is unlikely that the SLN effect is mediated by monomerization of PLB. However, this does not exclude the possibility that SLN could influence the inhibitory action of PLB, by binding allosterically to the same or to a different site. It should also be taken into account that, SLN is expressed at higher levels in fast-skeletal muscles of larger mammals, where PLB is absent, which readily suggests that SLN can regulate SERCA pump, independent of PLB. Future studies will be directed toward understanding the mechanism of SLN action on the SERCA pump in the absence of PLB.
In conclusion, we found that overexpression of SLN in mouse hearts decreases both SR calcium handling and cardiac contractility. The inhibitory effect of SLN can be relieved upon -adrenergic receptor stimulation and increased frequency, suggesting that it is a reversible inhibitor. The decreased contractility observed in SLN TG hearts is primarily caused by changes in the SLN/SERCA ratio, not alteration in PLB expression or phosphorylation status. Taken together our results suggest that SLN is a novel regulator of SERCA pump and plays an important role in cardiac physiology.
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FOOTNOTES |
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1 To whom correspondence should be addressed: Dept. of Physiology and Cell Biology, 304 Hamilton Hall, 1645 Neil Ave, The Ohio State University College of Medicine and Public Health, Columbus, OH 43210. Tel.: 614-292-2310; Fax: 614-292-4888; E-mail: periasamy.1{at}osu.edu.
2 The abbreviations used are: SR, sarco(endo)plasmic reticulum; SLN, sarcolipin; TG, transgenic; NTG, non-transgenic; SERCA, SR Ca2+ ATPase; PLB, phospholamban; RCC, rapid cooling contracture; RT, reverse transcriptase.
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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