Targeted Overexpression of Sarcolipin in the Mouse Heart Decreases Sarcoplasmic Reticulum Calcium Transport and Cardiac Contractility*

The role of sarcolipin (SLN) in cardiac physiology was critically evaluated by generating a transgenic (TG) mouse model in which the SLN to sarco(endoplasmic)reticulum (SR) Ca2+ ATPase (SERCA) ratio was increased in the ventricle. Overexpression of SLN decreases SR calcium transport function and results in decreased calcium transient amplitude and rate of relaxation. SLN TG hearts exhibit a significant decrease in rates of contraction and relaxation when assessed by ex vivo work-performing heart preparations. Similar results were also observed with muscle preparations and myocytes from SLN TG ventricles. Interestingly, the inhibitory effect of SLN was partially relieved upon high dose of isoproterenol treatment and stimulation at high frequency. Biochemical analyses show that an increase in SLN level does not affect PLB levels, monomer to pentamer ratio, or its phosphorylation status. No compensatory changes were seen in the expression of other calcium-handling proteins. These studies suggest that the SLN effect on SERCA pump is direct and is not mediated through increased monomerization of PLB or by a change in PLB phosphorylation status. We conclude that SLN is a novel regulator of SERCA pump activity, and its inhibitory effect can be reversed by β-adrenergic agonists.

The role of sarcolipin (SLN) in cardiac physiology was critically evaluated by generating a transgenic (TG) mouse model in which the SLN to sarco(endoplasmic)reticulum (SR) Ca 2؉ ATPase (SERCA) ratio was increased in the ventricle. Overexpression of SLN decreases SR calcium transport function and results in decreased calcium transient amplitude and rate of relaxation. SLN TG hearts exhibit a significant decrease in rates of contraction and relaxation when assessed by ex vivo work-performing heart preparations. Similar results were also observed with muscle preparations and myocytes from SLN TG ventricles. Interestingly, the inhibitory effect of SLN was partially relieved upon high dose of isoproterenol treatment and stimulation at high frequency. Biochemical analyses show that an increase in SLN level does not affect PLB levels, monomer to pentamer ratio, or its phosphorylation status. No compensatory changes were seen in the expression of other calcium-handling proteins. These studies suggest that the SLN effect on SERCA pump is direct and is not mediated through increased monomerization of PLB or by a change in PLB phosphorylation status. We conclude that SLN is a novel regulator of SERCA pump activity, and its inhibitory effect can be reversed by ␤-adrenergic agonists.
The sarco(endo)plasmic reticulum (SR) 2 Ca 2ϩ ATPase (SERCA) plays a dominant role in transporting Ca 2ϩ into the SR during the contraction-relaxation cycle of the heart. The rate and amount of Ca 2ϩ transported into the SR determines both the rate of muscle relaxation and the SR Ca 2ϩ load available for the next cycle of contraction (1)(2)(3)(4). It is well established that SERCA function is regulated by phospholamban (PLB), whose inhibitory effect is reversed by phosphorylation by protein kinase A and the calcium/calmodulin-dependent protein kinase (CAMKII) during adrenergic activation (5)(6)(7). Recent studies have shown that in addition to PLB, sarcolipin (SLN) could also play an important role in the regulation of SERCA pump activity (8 -12).
SLN is a 31-amino acid protein expressed in both cardiac and skeletal muscle (11,(13)(14)(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 ϳ50-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 Ca 2ϩ 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.

EXPERIMENTAL PROCEDURES
All experiments were performed in accordance with National Institutes of Health guidelines and approved by the Institutional Laboratory Animal Care and Use Committee at The Ohio State University.
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 plas-mid 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 (5Ј-GCCCACACCAGAAATGACAGA-3Ј) and the antisense primer specific for the 3Ј-end of SLN cDNA (5Ј-TCAGTATTGGTAGGACCTCA-3Ј). The copy number of the transgene was identified by Southern blot analysis of DNA samples from TG mice as described earlier (23).
To determine ␤-adrenergic agonist-mediated PLB phosphorylation, SLN TG hearts were perfused with isoproterenol in an isolated workperforming heart setup as described below. One set of hearts was freezeclamped after 30 min without isoproterenol, and the other set was treated with isoproterenol (1 M) 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 KP i , 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 g) 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 MgCl 2, 5 NaN 3 , 5 potassium oxalate, and 0.5 EGTA) and various concentrations of CaCl 2 to yield 0.03-3 mol/liter free Ca 2ϩ (containing 1 Ci/mol 45 Ca 2ϩ ). To obtain the maximal stimulation of SR Ca 2ϩ uptake, 1 M 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 Ca 2ϩ concentration required for half-maximal velocity of Ca 2ϩ uptake (EC 50 ) 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 CaCl 2 , 1.2 MgSO 4 , 0.5 Na-EDTA, 25 NaHCO 3 , 1.2 KH 2 PO 4 , and 11 glucose. All perfusion buffers were equilibrated with 95% O 2 plus 5% CO 2 , 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 (ϪdP/dt) and maximum (ϩdP/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 ϩdP/dt and ϪdP/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 Ϯ 21 m, 174 Ϯ 18, and 1401 Ϯ 78 (width ϫ thickness ϫ length in m, n ϭ 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 ([Ca 2ϩ ] 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).
In all the experiments performed, the parameters of developed force (F dev ) and diastolic force (F dia ) were determined and normalized to the cross-sectional area of the muscle. Additionally, the maximum speed of contraction (dF/dt max ) and relaxation (dF/dt min ) were measured. Preparations that did not exceed 10 mN/mm 2 sometime during the protocol or muscles that displayed excessive rundown of F dev (Ͼ10%/hour) were excluded. Multiple analyses of variance were used to determine significant differences between the interventions, with post-hoc t test when appropriate. A two-sided p value of Ͻ 0.05 was considered significant. n ϭ 6 -8 experiments are included in each protocol, and all values are expressed as the mean Ϯ S.E. unless stated otherwise.
Simultaneous Measurement of Ca 2ϩ 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%O 2 , 5%CO 2 ). The heart was then perfused with blenzyme solution (Roche Applied Science, 37°C, bubbled with 95%O 2 , 5%CO 2 ). 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 M Ca 2ϩ were added. After gravity-settled, cells were resuspended in MEM containing 200 M Ca 2ϩ .
Myocytes were loaded at 22°C with Fluo-4 AM (10 M, 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 MgCl 2 , 1 CaCl 2 , 10 glucose, and 5 HEPES (pH 7.4). [Ca 2ϩ ] i was measured by Fluo-4 epifluorescence with excitation at 480 Ϯ 20 nm and emission at 535 Ϯ 25 nm. The illumination field was restricted to a small spot to get emission from a single cell. Data were expressed as F/F o , where F is the fluorescence intensity and F o 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 Ca 2ϩ 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 Ϯ S.E. Statistical significance was estimated by a paired Student's t test. A value of p Ͻ 0.05 was considered statistically significant.

RESULTS
Generation of SLN TG Mice-To determine how SLN regulates cardiac function and SR calcium transport, we overexpressed FLAGtagged mouse SLN cDNA under the control of the ␣-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 F 0 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 expres- 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 Ϯ 2.3% to 169.47 Ϯ 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 (DHPR␣2), and triadin to determine changes in Ca 2ϩ release and entry mechanisms. As shown in Fig. 2B, SLN overexpression did not affect the RyR, DHPR␣2, 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.
Basal Phosphorylation of PLB Is Not Affected by SLN Overexpression-In a recent study, Asahi et al. (12) reported that cardiac-specific overexpression of SLN resulted in decreased basal phosphorylation of PLB and an increase in the monomer to pentamer ratio. To test whether overexpression of SLN resulted in monomerization of PLB and decreased phosphorylation, Western blot analysis was carried out using appropriate antibodies. Our results (Fig. 3) clearly show that the monomer to pentamer ratio was not altered in the TG ventricle. Further, the basal phosphorylation of PLB at serine 16 (Ser 16 ) and threonine 17 (Thr 17 ) was not different between the TG and NTG ventricles (Fig. 3). These results contradict a previous report (12) and show that SLN overexpression did not alter either PLB basal phosphorylation or the monomer to pentamer ratio.
SLN Overexpression Decreases the Apparent Ca 2ϩ Affinity and Rate of Ca 2ϩ 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 Ca 2ϩ affinity in SLN TG ventricles (Fig. 4) 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 (ϩdP/dt) and relaxation (ϪdP/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). FIGURE 2. Quantification of SR calcium-handling proteins and sarcolemmal calcium transporters, NCX and PMCA. Different SDS-PAGE gel concentrations (5% for RyR, NCX, and PMCA, 8% for SERCA and CSQ, 10% for DHPR␣2 and triadin, and 14% for PLB) were used to resolve total ventricular homogenates from NTG and TG mice and immunoprobed with specific antibodies. We next examined the force-frequency relationship to determine the frequency-dependent contractile reserve in the SLN TG heart. For these experiments, TG and NTG hearts were paced with frequencies from 4 to 11 Hz, and the rates of contraction (ϩdP/dt) and relaxations were determined at multiple intervals. As shown in Fig. 5A both SLN TG and NTG control hearts demonstrated the flattened force-frequency dependence (no ascending limb). However in mice overexpressing SLN, the force-frequency relation was shifted to the right, indicating that contractility is decreased in the SLN TG hearts. However, at very high stimulation frequencies (11 Hz), rates of contraction and relaxation of SLN TG hearts were not significantly different from NTG controls.
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 ϩdP/dt and ϪdP/dt were restored to the level of control hearts after infusion of high doses of isoproterenol.
The phosphorylation status of PLB at Ser 16 and Thr 17 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 Ca 2ϩ load. At a baseline frequency of 4 Hz, TG mice showed an RCC amplitude of 7.29 Ϯ 1.76 mN/mm 2 compared with 11.65 Ϯ 1.62 mN/mm 2 in NTG mice (NS, p ϭ 0.12). At a stimulation of 12 Hz, TG mice showed a decreased RCC amplitude of 6.13 Ϯ 1.56 mN/mm 2 (n ϭ 6), compared with the NTG littermates (11.03 Ϯ 1.19 mN/mm 2 , n ϭ 7, p Ͻ 0.05). These data suggest that overexpression of SLN decreases SR calcium load.
Overexpression of SLN Decreases Ca 2ϩ Transient Amplitude and Slows Relaxation in Ventricular Myocytes-The effect of SLN overexpression on myocyte Ca 2ϩ handling was studied using isolated ventricular myocytes from TG and NTG hearts (Fig. 7). Myocytes from SLN TG ventricles showed a decrease in Ca 2ϩ transient amplitude by 57% (Fig. 7B) and significantly prolonged the rate of relaxation (NTG, 163 Ϯ 14 ms versus TG, 255 Ϯ 16 ms). In addition, shortening data paralleled the Ca 2ϩ 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 Ca 2ϩ transient amplitude and relaxation (0.2 Hz, NTG: 429 Ϯ 31 ms versus SLN TG, 520 Ϯ 43 ms, p Ͻ 0.05). However, at higher frequencies, the relaxation times were similar in both groups (2 Hz, NTG: 170 Ϯ 7 ms versus SLN TG, 171 Ϯ 4 ms, p ϭ NS). In addition, Ca 2ϩ transient amplitude and shortening at lower frequencies was significantly reduced in TG versus NTG myocytes, but similar at higher frequencies (data not shown).

DISCUSSION
To better define the role of SLN in cardiac physiology, we chose to increase the ratio of SLN to SERCA2a in the ventricle (because it is naturally low) and study how an increase in SLN affects calcium homeostasis and heart function. The major finding of our study is that overexpression of SLN in the ventricle resulted in decreased SERCA pump affinity for calcium, Ca 2ϩ transient amplitude and shortening, and slowed relaxation. More importantly, our data indicate that the inhibitory effect of SLN can be reversed by the ␤-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 FIGURE 4. Calcium uptake function in NTG and SLN TG mice. Ca 2ϩ uptake assays were performed using whole heart homogenates from 24-week-old mice (n ϭ 4 for each group). The V max of Ca 2ϩ uptake was obtained at pCa 6.0 in both groups. mouse hearts reduced the apparent Ca 2ϩ affinity of the SERCA pump.
In vivo measurements of cardiac function showed significant decreases in ϩdP/dt and ϪdP/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 hypertro-phy 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). The finding that overexpression of SLN results in decreased pump affinity for Ca 2ϩ and depressed SR Ca 2ϩ load (as measured by RCC experiments) suggests that SLN is an inhibitor of the SERCA pump. This is further supported by decreased Ca 2ϩ transient amplitudes and slowed rates of relaxation in isolated myocytes. Consistent with the myocyte studies, both ϩdP/dt and ϪdP/dt were found to be significantly decreased in the SLN TG heart as assessed by the hemodynamic measurements. Similar observations have also been made in muscle FIGURE 5. Work-performing heart preparations. A, rate of contraction (ϩdP/dt) and relaxation (ϪdP/dt) in response to increase in force. Data were derived from 6 NTG and 5 SLN TG mice. B, cumulative dose-response to isoproterenol in isolated work-performing hearts. Data were derived from 5 NTG and 4 SLN TG mice. * indicates significant difference between groups, p Ͻ 0.05. C, Western blot analysis of PLB phosphorylation in isolated work-performing hearts before (ϪIso) and after (ϩIso) isoproterenol (1 M) perfusion. Tissue samples were processed for total protein extraction and immunoprobed with Ser 16 -or Thr 17 -specific PLB antibodies. FIGURE 6. Rate of contraction (؉dF/dt) and relaxation (؊dF/dt) in muscle preparations from SLN TG and NTG hearts. A, force-frequency response of muscle contraction and relaxation at 6 and 14 Hz. B, isoproterenol (1M) response of muscle contraction from SLN TG and NTG hearts. * indicates significant difference between groups (p Ͻ 0.05). NTG, n ϭ 6 and SLN TG, n ϭ 8.
preparations. These results corroborate nicely with our recent report on myocyte contractility and Ca 2ϩ transients using adenoviral-mediated SLN overexpression in rat ventricular myocytes (11). These functional changes, however, are not sufficient to cause any cardiac pathology. It is quite likely that some of these functional changes could be compensated by mechanisms yet to be determined.
An interesting finding of this study is that the inhibitory effect of SLN on contractile function could be reversed by treatment with isoproterenol and at high frequency. MacLennan and co-workers (12) also reported similar findings in SLN TG hearts, where isoproterenol restored contractile function. They reported that basal PLB phosphorylation was decreased in SLN TG hearts and upon adrenergic stimulation, PLB phosphorylation was restored to the same extent as control hearts. The authors interpreted that enhanced PLB phosphorylation (which may result in dissociation of SLN from PLB) could be the mechanism for restoration of function in SLN TG hearts during ␤-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 (Thr 5 ) 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 Thr 5 to Ala leads to a slight gain in inhibitory function (18) further supporting the idea that phosphorylation of Thr 5 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 Thr 5 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). Phos-phorylation 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.