Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 278, No. 11, Issue of March 14, pp. 9570 –9575, 2003 Printed in U.S.A. Atrial Chamber-specific Expression of Sarcolipin Is Regulated during Development and Hypertrophic Remodeling* Received for publication, December 23, 2002 Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M213132200 Susumu Minamisawa‡§¶, Yibin Wang储, Ju Chen**, Yoshihiro Ishikawa§‡‡, Kenneth R. Chien**, and Rumiko Matsuoka‡¶§§ From the ‡Department of Pediatric Cardiology and the §§Division of Genomic Medicine, Institute of Advanced Biomedical Engineering and Science, Graduate School of Medicine, Tokyo Women’s Medical University, Tokyo 162-8666, Japan, the §Department of Physiology, Yokohama City University School of Medicine, Yokohama, 236-0004 Japan, the 储Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201, the **Institute of Molecular Medicine, University of California at San Diego, La Jolla, California 92093, and the ‡‡Department of Cell Biology and Molecular Medicine, New Jersey Medical School, Newark, New Jersey 07103 Intracellular Ca2ⴙ regulation is critical in the normal cardiac function and development of pathologic hearts. Phospholamban, an endogenous inhibitor of sarcoplasmic reticulum Ca2ⴙ ATPase in the sarcoplasmic reticulum, plays an important role in Ca2ⴙ cycling in heart. Recently, sarcolipin has been identified as having a similar function as phospholamban in skeletal muscle. Because phospholamban is differentially expressed in atrial and ventricular myocardia and its expression is often altered in diseased hearts, we investigated the cardiac chamber specificity of sarcolipin expression and its regulation during development and hypertrophic remodeling. Northern blot analysis revealed that the expression of mouse sarcolipin mRNA was most abundant in the atria and was undetectable in the ventricles, indicating an atrial chamber-specific expression pattern. Atrial chamber-specific expression of sarcolipin mRNA was increased during development. These findings were confirmed by in situ hybridization studies. In addition, sarcolipin expression was down-regulated in the atria of hypertrophic heart when induced by ventricular specific overexpression of the activated H-ras gene. In humans, sarcolipin mRNA was also expressed in the atria but not detected in the ventricles, although sarcolipin expression was most abundant in skeletal muscle. Taken together, sarcolipin is likely to be an atrial chamber-specific regulator of Ca2ⴙ cycling in heart. Sarcolipin (SLN)1 is a 31-amino acid proteolipid in the sarcoplasmic reticulum (SR) (1) and is shown to be expressed most abundantly in fast twitch skeletal muscle, less abundantly in slow twitch skeletal muscle, and even less in human and rabbit * This work was supported in part by an open research grant from the Japan Research Promotion Society for Cardiovascular Diseases (2001). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ¶ To whom correspondence may be addressed: Dept. of Pediatric Cardiology, The Heart Institute of Japan, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. Tel.: 81-33353-8111, ext. 24064; Fax: 81-33352-3088; E-mail: sminamis@ med.yokohama-cu.ac.jp or [email protected]. 1 The abbreviations used are: SLN, sarcolipin; SR, sarcoplasmic reticulum; SERCA, SR Ca2 ATPase; PLN, phospholamban; GFP, green fluorescent protein; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; ANF, atrial natriuretic factor; BNP, brain natriuretic peptide. cardiac muscle (2). This tissue distribution corresponds to that of the fast twitch skeletal muscle SR Ca2⫹ ATPase (SERCA1), and SLN interacts with SERCA1 to modulate its activity (3, 4). By contrast, phospholamban (PLN), an integral membrane protein in the SR, is expressed abundantly in cardiac and slow twitch skeletal muscle wherein SERCA2a is the predominant SERCA isoform (5). Thus, SLN expression is complementary to PLN expression. The structure and protein sequence of SLN are similar to those of PLN (2). Therefore, SLN and PLN may belong to the same family, and SLN is likely to be an analog of PLN in skeletal muscle. PLN is an endogenous inhibitor of SERCA2 and plays a central role in regulating cardiac contractility and relaxation (6, 7). Studies using gene-targeted mice have revealed that disruption of the PLN gene can prevent the progression of cardiomyopathy (8, 9), suggesting that PLN plays a critical role in the development of heart failure. The expression of PLN is regulated by developmental, hormonal, and hemodynamic changes (7). In the heart, PLN is expressed predominantly in the ventricles and, to a lesser extent, in the atria (10). The different levels of PLN expression in the heart are, in part, responsible for the differences in the contractile properties between the atria and the ventricles. In contrast to PLN, the role of SLN in the heart remains unknown, even though SLN may play an important role in skeletal muscle contraction (11). Because SLN expression is complementary to PLN expression, we hypothesize that SLN expression in the heart is higher in the atria and is regulated in a developmental stage-specific manner and/or by cardiac stress. In addition, Asahi et al. (4) demonstrated recently that SLN inhibited polymerization of PLN, resulting in the superinhibition of SERCA1 and SERCA2a. Therefore, characterizing the expression pattern of SLN is important for establishing the potential modulatory role in cardiac function. In the present study, we examined the effects of development and cardiac hypertrophy on the expression of SLN mRNA in heart. We found that the expression of SLN mRNA was detected selectively in the atria. Furthermore, SLN was up-regulated during development and down-regulated by cardiac hypertrophy. SLN mRNA remained undetectable in the ventricles during development or in the hypertrophic state, indicating that the atrial chamber-specificity of SLN was conserved. Taken together, we propose that SLN may serve an important regulator of Ca2⫹ cycling in the atrium and may be an important factor in differentiating the contractile properties of the atria from those of the ventricles. 9570 This paper is available on line at http://www.jbc.org Atrial Chamber-specific Expression of Sarcolipin 9571 TABLE I Primers Gene Mouse SLN Human SLN Mouse SERCA1 EMBL/GenBank™ accession number NM_025540 U96094 NM_007504 Forward Reverse 5⬘-CTGAGGTCCTTGGTAGCCTG-3⬘ 5⬘-GGTGTGTCAGGCATTGTGAG-3⬘ 5⬘-GAGGTGAGGACAAGCCAGAG-3⬘ 5⬘-GTCTCAGGGCATAGAGCAGG-3⬘ 5⬘-TGCTCGGAACTATCTGGAGG-3⬘ 5⬘-GAGTTCGGGAAGGGGATTTA-3⬘ Size of PCR product 331-bp 237-bp 258-bp FIG. 1. Atrial chamber-specific expression of sarcolipin mRNA. A, northern blot analysis for the various mouse tissues. Using the full-length SLN cDNA probe, a single transcript of 0.9 kb was expressed most abundantly in the atria of the heart, less abundantly in esophageal muscle, and least abundantly in skeletal muscle and bladder. No hybridized signal was detected in the ventricles. A small amount of SERCA1 transcript was detected in the atria. There was robust SERCA1 expression in mouse smooth muscle. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for internal control. B, in situ hybridization analysis using an antisense SLN RNA probe. SLN mRNA was expressed restrictedly in the atria but not in the ventricles. The localization of SLN transcript was distributed uniformly in both the right and left atria. C, a high magnification image of the lesion indicated as a rectangle in panel B. EDL, extensor digitorum longus muscle; LV, left ventricle; RV, right ventricle; LA, left atrium; RA, right atrium. Bar, 1 mm (B). EXPERIMENTAL PROCEDURES Animals and Tissue Samples—ICR mice obtained from Clea Japan Inc. (Tokyo, Japan) were inbred at Tokyo Women’s Medical University. Embryos from ICR mice were obtained from timed pregnant animals. The transgenic model of cardiac hypertrophy was established by ventricular specific expression of activated H-ras transgene. In this study, we applied the same “gene-switch” transgenic strategy as reported previously (12). Briefly, a green fluorescence protein (GFP) coding sequence and poly(A) regions were cloned from pEGFP-N1 (Invitrogen) and inserted between the two loxP sequences of the pUC1015loxlox vector (a gift from Dr. Jamy Marth, University of California, San Diego, CA) to generate the pfloxGFP vector. The XbaI/SmaI fragment from pfloxGFP, containing the GFP-poly(A) sequences flanked by two copies of loxP, was then inserted at the HindIII site of the pMHC vector behind the 5.5 kilobase mouse ␣-MHC promoter (a gift from Dr. Jeffery Robbins, University of Cincinnati) to generate the pMHC-floxGFP vector. The cDNA fragment coding for the activated H-ras (V15) mutant was then inserted at the EcoRV site behind the loxP-GFP-loxP fragment to generate the final pMHC-floxRas vector. Transgenic mice (floxRas) were generated by established intranuclear injection methods using a KpnI/SstI fragment digested from the pMHC-floxRas vector. The GFP marker gene was expressed in both the atria and ventricles. The floxRas mice were crossed with MLC-2v/Cre mice in which the Cre enzyme was only expressed in the ventricular muscle cells. The GFP expression cassette was floxed out, and the activated H-ras mutant was led to be expressed in the ventricles of double transgenic mice. The effect of the H-ras mutant on cardiac hypertrophy has been validated (13). Human tissues were obtained from specimens at autopsy. Informed consent was obtained from the patients and/or their families. Total RNA Isolation—Total RNA was isolated from various tissues using TRIzol reagent (Invitrogen) as recommended by the manufacturer. For the developmental study, atrial and left ventricular tissues from embryos and neonates were pooled. The isolated RNA was quantified by spectrophotometry. cDNA and RNA Probes—To obtain the mouse and human full-length SLN cDNAs and mouse partial-length SERCA1 cDNA, a set of primers was designed based on the reported sequence (Table I). The mouse SERCA1 cDNA included the end of the coding sequence and 3⬘-untranslated region. Reverse transcription PCR was performed using the Superscript preamplification system (Invitrogen) as recommended by the manufacturer. The reaction of PCR was set to denature at 94 °C for 30 s, anneal at 55 °C for 30 s, and extend at 72 °C for 45 s for 30 cycles. The obtained PCR fragments were subcloned in pCR II vector (Invitrogen). The antisense mouse SLN digoxygenin-labeled RNA probe was created from 1 g of HindIII-linearized mouse SLN pCRII vector using T3 RNA polymerase. Mouse SERCA2a and PLN cDNA probes were kindly provided by Dr. Wolfgang H. Dillmann (University of California, San Diego, CA) and Dr. Evangelia G. Kranias (University of Cincinnati College of Medicine), respectively. We confirmed the nucleotide sequences of these 9572 Atrial Chamber-specific Expression of Sarcolipin probes compared with the reported sequences by direct DNA sequencing. Northern Blot Analysis—Northern blot analysis was performed as described previously with modification (14). Total RNA was electrophoresed on a 1% formaldehyde-agarose gel and transferred onto Nytran membranes (Schleicher & Schuell). Blots were hybridized at 68 °C with radiolabeled probes in Quickhyb solution (Stratagene, La Jolla, CA) as recommended by the manufacturer. Membranes were washed under high stringency conditions (0.1⫻ SSC, 0.1% SDS) at 60 °C. In Situ Hybridization—In situ hybridization was performed as described previously (15). Paraffin sections were prepared using a standard protocol. SERCA Activity—Microsomes enriched in SR membranes were obtained as described previously (16). SR Ca2⫹ ATPase activity was measured as described previously with modification (17). Using 5 g of SR protein, the reaction was carried out at 37 °C in the reaction medium containing 30 mM TES, 100 mM KCl, 5 mM NaN3, 5 mM MgCl2, 0.5 mM EGTA, and 4 mM ATP with or without 0.5 CaCl2. Data are expressed as mean ⫾ S.E. Statistical significance was analyzed using Student’s unpaired t test. RESULTS SLN mRNA Was Expressed Abundantly in the Murine Atria—The distribution of SLN mRNA in various mouse tissues was analyzed by Northern blot using a PCR-amplified cDNA probe as described under “Experimental Procedures.” A single 0.9-kb transcript was detected. Specifically, SLN mRNA expression was most abundant in the atria of the heart, less abundant in esophageal muscle, and least abundant in skeletal muscle and bladder (Fig. 1A). In murine myocardium, the expression of SLN mRNA was restricted to the atria and was not present in the ventricles. In skeletal muscle, the similar level of SLN mRNA expression was detected in fast twitch skeletal muscle (extensor digitorum longus muscle) and slow twitch skeletal muscle (soleus muscle). In smooth muscle, SLN mRNA expression was much greater in esophagus than in bladder. SLN mRNA was not detected in brain, kidney, liver, spleen, thymus, and lung (liver, spleen, thymus, and lung, data not shown) by Northern blot analysis. In situ hybridization analysis using an antisense SLN RNA probe also demonstrated that SLN mRNA was expressed only in the atria and not in the ventricles of the heart (Fig. 1B). The localization of SLN transcript was distributed uniformly in both right and left atria. We also examined the expression of SERCA1 mRNA in the mouse heart, because SLN is believed to interact with SERCA1. As shown in Fig. 1A, a very small amount of the SERCA1 transcript was detected in the atria. The level of SERCA2 mRNA in the atria was comparable with that in the ventricles. At the protein level, only SERCA2a but not SERCA1 was detected in the atria, using SERCA isoform-specific antibodies (data not shown; SERCA1 antibody (MA3–912) and SERCA2 antibody (MA3–919) were from Affinity Bioreagents, Inc.). There was abundant SERCA1 expression in mouse smooth muscle. SERCA1 mRNA levels were similar in esophagus and bladder. The tissue distribution of PLN and SERCA2a was consistent with previous reports (18, 19). SR Ca2⫹ ATPase activity in the atria (n ⫽ 5) was significantly higher than that in the ventricles (n ⫽ 7) (1573 ⫾ 85 nmol/mg of protein/min and 348 ⫾ 35 nmol/mg of protein/min, respectively) in adult murine hearts. SLN Was Developmentally Up-regulated in the Atria—The developmental changes of SLN and SERCA1 mRNA in the atria were also determined by Northern blot analysis. Fig. 2A demonstrates that the expression of SLN and SERCA1 mRNA was increased over time in the atria and not detected in the ventricles at any of the developmental stages. Interestingly, the expression of SERCA1 mRNA became very low in the atria in older mice (2 years old), suggesting that SERCA1 expression is down-regulated in the atria with aging. The expression of ANF transcript FIG. 2. Developmental up-regulation of sarcolipin mRNA in the atria. A, the developmental change in the expression of SLN, SERCA1, and ANF mRNA in the atrial and ventricular myocardium. Northern blot analysis revealed that these transcripts are up-regulated during development in the atria. The expression of ANF transcript was also detected in the ventricles of embryos at embryonic day 17.5 and in older mice (at 2 years old). B, the developmental changes in the atrial expression of SLN and SERCA1 mRNA in detail. No SLN transcript was detected in embryos at embryonic day (ed) 10.5 but became detectable in the atria at embryonic day 12.5. After embryonic day 16.5, the expression of SLN mRNA was abruptly increased. was also increased over time in the atria. In contrast to SLN, ANF transcripts were detected in the ventricles of embryos at embryonic day 17.5 and at 2 years old of age. We further examined the developmental changes of SLN and SERCA1 mRNA expression in detail. No SLN transcript was found in embryos at embryonic day 10.5 but became detectable in the atria at embryonic day 12.5 (Fig. 2B). After embryonic day 16.5, the expression of SLN mRNA was abruptly increased in the atria. These changes were confirmed by in situ hybridization studies using embryos at specific developmental stages. At embryonic day 9.5 no SLN transcript was detected (data not shown), whereas at embryonic day 12.5 SLN mRNA was present at a low level in the atria and expressed abundantly in the progenitors of skeletal muscle (Fig. 3). SERCA1 mRNA was expressed weakly in the atria at embryonic day 10.5 and 12.5 but was not detectable after embryonic day 16.5 (Fig. 2B). The SERCA1 transcript was detected again at 21 days after birth. Thus, the developmental changes in SLN and SERCA1 transcripts were not exactly coordinated. SLN and SERCA1 mRNA Were Down-regulated in the Atria in Hypertrophic Hearts—The transgenic mice overexpressing activated H-ras developed severe concentric ventricular hypertrophy with concomitant obstruction of forward blood flow from the atria to the ventricles, resulting in marked enlargement of atrial chambers (Fig. 4). The levels of SLN, PLN, SERCA1 and SERCA2a mRNA were all decreased in the atria of the transgenic mice at 3 months old (Fig. 5). Although ANF and BNP Atrial Chamber-specific Expression of Sarcolipin 9573 FIG. 3. In situ hybridization analysis of sarcolipin mRNA in an embryo at embryonic day 12.5. Using an antisense SLN riboprobe, SLN mRNA was detected at low levels in the atria and was much higher in the progenitors of skeletal muscle. A, atrium; L, liver; T, tongue; V, ventricle. Bar, 1 mm. FIG. 4. Marked ventricular hypertrophy and dilated atria in Ras transgenic mice. The activated H-ras transgenic mice developed severe concentric ventricular hypertrophy and marked enlargement of atrial chambers (A, B, and C, non-transgenic mice; D, E, F, and H, activated H-ras transgenic mice). Panels A and D are the cross-sections of the ventricles. Severe concentric ventricular hypertrophy was detected in the activated H-ras transgenic mice (D). Panel E is a high magnification image of the left ventricle of the activated H-ras transgenic mice demonstrating disorganized myofibrils. Organized thrombi were found frequently in the atria of the activated H-ras transgenic mice (panels F and H). Panel H is the frontal section of a H-ras transgenic heart. transcripts were increased in both the atria and ventricles of the transgenic mice, SLN mRNA remained undetectable in the hypertrophic ventricles. SLN mRNA Was Also Expressed Specifically in the Human Atria—Although a previous study showed that SLN expression was low in human heart, its chamber-specific localization has 9574 Atrial Chamber-specific Expression of Sarcolipin FIG. 5. Down-regulation of sarcolipin and SERCA1 mRNA in H-ras transgenic mice. The representative Northern blot analysis of the activated H-ras transgenic mice. The expressions of SLN, PLN, SERCA1, and SERCA2a mRNA were all down-regulated in the atria of the H-ras transgenic mice at 3 months old. ANF and BNP were induced in the atria and the ventricles of the H-ras transgenic mice. RasTG, the activated Hras transgenic mice; NTG, non-transgenic mice as control. GAPDH, glyceraldehyde3-phosphate dehydrogenase. remained undetermined. We found that SLN mRNA was expressed abundantly in human atria and was undetectable in ventricles (Fig. 6). In contrast to mouse, the expression of SLN in the atria was lower than in skeletal muscle in human. Using the mouse SERCA1 cDNA probe that has 75% homology with human SERCA1, no hybridization signal was detected in the human atria, even under low stringent conditions. DISCUSSION The present study revealed that SLN mRNA was expressed abundantly in the murine and human atria but was not detected in the murine and human ventricles by Northern blot analysis. In mice, the expression of SLN mRNA was most abundant in the atria, and it was lower in skeletal muscle than in the atria and smooth muscle. By contrast, in humans the expression of SLN mRNA was weaker in the atria than that in skeletal muscle. Previous studies have demonstrated the predominant expression of SLN mRNA in human and rabbit fast skeletal muscle (2) and the very weak expression of SLN mRNA in rat extensor digitorum longus (EDL) muscle (20). These data are consistent with the findings in the present study, suggesting that the tissue distribution of sarcolipin mRNA is regulated differently between humans and rodents. The fact that the nucleotide sequence of SLN cDNA is not well conserved between mice and humans and that their polyadenylation signal sequences are different (2) may account for the different tissue distribution between rodents and humans. Importantly, the atrial chamber-specific expression of SLN was developmentally up-regulated, and it was down-regulated in hypertrophic remodeling at the transcriptional level. These changes are similar to those of PLN in the ventricles (21) (22). No ventricular SLN expression was detected at any stage of development or cardiac hypertrophy. We did not investigate the protein levels of SLN, because a SLN antibody was not readily available. However, a previous study (2) demonstrated that changes in the SLN levels of mRNA and protein are proportional, although the magnitude of the changes in SLN protein was greater than that in SLN mRNA in the skeletal muscle. Therefore, it is likely that the SLN protein is also expressed abundantly in the murine atria. PLN decreases the apparent affinity of SERCA2a for Ca2⫹ and inhibits Ca2⫹ uptake into the SR. The expression level of FIG. 6. The high expression of sarcolipin mRNA in the human atria. Abundant SLN expression was also detected in the human atria but not in the ventricles. The expression was most abundant in skeletal muscle. Using a mouse SERCA1 cDNA probe, no hybrydization signal was detected in the human atria. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. PLN directly affects cardiac contractility and relaxation in the heart (23, 24). SLN also inhibits Ca2⫹ uptake at low Ca2⫹ concentrations like PLN but enhances Ca2⫹ uptake at high Ca2⫹ concentrations (3, 25). Therefore, the atrial chamberspecific expression of SLN may be related to functional differences between the atria and ventricles, and the abundant ex- Atrial Chamber-specific Expression of Sarcolipin pression of SLN may play an important role in the regulation of Ca2⫹ cycling in the atrial myocardium. The coordinated contraction of the atria with the ventricles is essential for normal cardiac output. However, the physiological characteristics are quite different between atrial and ventricular myocardium. For examples, muscle contraction time, measured as a time to peak tension, and the duration of the myocyte Ca2⫹ transients are shorter in atrial muscle compared with ventricular muscle. In the present study, we also found that SR Ca2⫹ ATPase activity is significantly higher in the atria than in the ventricles. This may be due to different levels of PLN expression between atria and ventricles (10). In addition to the lower expression of PLN, SLN may also contribute to the chamber-specific physiological properties of the atria. Furthermore, we found that SERCA1 mRNA was expressed weakly in the mouse atria and that its expression was also regulated during development and hypertrophic remodeling. However, it is unlikely that SERCA1 contributes to the higher ATPase activity in the atria, because SERCA2a is the predominant isoform, and the SERCA1 protein was not detected in the atria. In skeletal muscle, SLN expression is regulated by chronic low frequency stimulation (3) or corticosteroids (20). Odermatt et al. (3) suggested that reduced SLN expression associated with the decrease in SERCA1 activity represented an early functional adaptation to chronic low frequency stimulation. They proposed that SLN provides a constant stimulation of SERCA Vmax to increase the activity of SERCA. Thus, the abundant expression of SLN with an increase in the activity of SERCA and faster contraction in the atria is consistent with this model. However, these investigators demonstrated recently that SLN inhibited both SERCA1 and SERCA2a activities in human embryonic kidney 293 cells (4) and in in vivo slow skeletal muscle (11). Moreover, they suggested that SLN binds directly to PLN and increases the formation of PLN monomers that inhibits SERCA activity (4). If these data are true in the in vivo heart, overexpression of SLN should result in the depression of SERCA activity and slow muscle contraction. Further studies using transgenic mice overexpressing SLN in heart are needed to address these issues. Using a Ras transgenic mouse model for hypertrophy, we demonstrated that SLN mRNA was down-regulated in cardiac hypertrophy. Isolated cardiomyocytes from Ras transgenic mice displayed reduced contraction and prolonged Ca2⫹ transients compared with wild-type myocytes2 despite the reduction of PLN expression. Other studies also demonstrated that Ca2⫹ cycling in the atria is impaired in cardiac hypertrophy or chronic atrial stimulation such as atrial fibrillation. Therefore, decreases in the expression of SLN, SERCA1, and SERCA2 may result in the reduction of Ca2⫹ uptake in the atria and may play a more important role in maintaining the atrial function than the level of PLN expression. Another important finding in the present study is that SLN mRNA was not expressed in the ventricular myocardium during any stage of development or in a cardiac hypertrophic state, indicating that SLN is an atrial chamber-specific molecular 2 S. Miamisawa, Y. Wang, and K. R. Chen, unpublished observations. 9575 marker. In contrast to SLN, other atrial chamber-specific molecular markers such as ANF and atrial type of myosin light chain 1 are induced in ventricles by cardiac stresses. Therefore, a putative promoter/enhancer gene fragment of SLN may be useful for atrial chamber-specific gene targeting or gene delivery. In conclusion, we found that SLN, a counterpart of PLN, was expressed specifically in the atrial myocardium. The atrial chamber-specific expression of SLN was up-regulated during development and down-regulated in hypertrophic remodeling. The present study suggests that SLN is an important regulator of Ca2⫹ cycling in the atrium. Acknowledgments—We thank Dr. Tomoyuki Nakamura and Dr. Yoji Sato for stimulating discussion, Dr. Paul Grossfeld for critical reading of the manuscript, Ms. Barbara Levene for language editing of the manuscript, and Ms. Keiko Komatsu for preparation of paraffin sections. REFERENCES 1. Wawrzynow, A., Theibert, J. L., Murphy, C., Jona, I., Martonosi, A., and Collins, J. H. (1992) Arch Biochem. Biophys. 298, 620 – 623 2. Odermatt, A., Taschner, P. E., Scherer, S. W., Beatty, B., Khanna, V. K., Cornblath, D. R., Chaudhry, V., Yee, W. C., Schrank, B., Karpati, G., Breuning, M. H., Knoers, N., and MacLennan, D. H. (1997) Genomics 45, 541–553 3. Odermatt, A., Becker, S., Khanna, V. K., Kurzydlowski, K., Leisner, E., Pette, D., and MacLennan, D. H. (1998) J. Biol. Chem. 273, 12360 –12369 4. Asahi, M., Kurzydlowski, K., Tada, M., and MacLennan, D. H. (2002) J. Biol. Chem. 277, 26725–26728 5. Tada, M., and Toyofuku, T. (1996) J. Card. Fail. 2, Suppl. 4, S77–S85 6. Luo, W., Grupp, I. L., Harrer, J., Ponniah, S., Grupp, G., Duffy, J. J., Doetschman, T., and Kranias, E. G. (1994) Circ. Res. 75, 401– 409 7. Koss, K. L., and Kranias, E. G. (1996) Circ. Res. 79, 1059 –1063 8. Minamisawa, S., Hoshijima, M., Chu, G., Ward, C. A., Frank, K., Gu, Y., Martone, M. E., Wang, Y., Ross, J., Jr., Kranias, E. G., Giles, W. R., and Chien, K. R. (1999) Cell 99, 313–322 9. Sato, Y., Kiriazis, H., Yatani, A., Schmidt, A. G., Hahn, H., Ferguson, D. G., Sako, H., Mitarai, S., Honda, R., Mesnard-Rouiller, L., Frank, K. F., Beyermann, B., Wu, G., Fujimori, K., Dorn, G. W., II, and Kranias, E. G. (2001) J. Biol. Chem. 276, 9392–9399 10. Koss, K. L., Ponniah, S., Jones, W. K., Grupp, I. L., and Kranias, E. G. (1995) Circ. Res. 77, 342–353 11. Tupling, A. R., Asahi, M., MacLennan, D. H., Kurzydlowski, K., and Tada, M. (2002) J. Biol. Chem. 277, 44740 – 44746 12. Liao, P., Georgakopoulos, D., Kovacs, A., Zheng, M., Lerner, D., Pu, H., Saffitz, J., Chien, K., Xiao, R. P., Kass, D. A., and Wang, Y. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 12283–12288 13. Hunter, J. J., Tanaka, N., Rockman, H. A., Ross, J., Jr., and Chien, K. R. (1995) J. Biol. Chem. 270, 23173–23178 14. Minamisawa, S., Gu, Y., Ross, J., Jr., Chien, K. R., and Chen, J. (1999) J. Biol. Chem. 274, 10066 –10070 15. Machida, S., Matsuoka, R., Noda, S., Hiratsuka, E., Takagaki, Y., Oana, S., Furutani, Y., Nakajima, H., Takao, A., and Momma, K. (2000) Dev. Dyn. 217, 37– 49 16. Edes, I., and Kranias, E. G. (1990) Circ. Res. 67, 394 – 400 17. Nakanishi, T., and Jarmakani, J. M. (1984) Am. J. Physiol. 246, H61–H625 18. Grover, A. K., and Khan, I. (1992) Cell Calcium 13, 9 –17 19. Tada, M., Yabuki, M., and Toyofuku, T. (1998) Ann. N. Y. Acad. Sci. 853, 116 –129 20. Gayan-Ramirez, G., Vanzeir, L., Wuytack, F., and Decramer, M. (2000) J. Physiol. 524, 387–397 21. Ganim, J. R., Luo, W., Ponniah, S., Grupp, I., Kim, H. W., Ferguson, D. G., Kadambi, V., Neumann, J. C., Doetschman, T., and Kranias, E. G. (1992) Circ. Res. 71, 1021–1030 22. Moorman, A. F., Vermeulen, J. L., Koban, M. U., Schwartz, K., Lamers, W. H., and Boheler, K. R. (1995) Circ. Res. 76, 616 – 625 23. Luo, W., Wolska, B. M., Grupp, I. L., Harrer, J. M., Haghighi, K., Ferguson, D. G., Slack, J. P., Grupp, G., Doetschman, T., Solaro, R. J., and Kranias, E. G. (1996) Circ. Res. 78, 839 – 847 24. Dash, R., Kadambi, V., Schmidt, A. G., Tepe, N. M., Biniakiewicz, D., Gerst, M. J., Canning, A. M., Abraham, W. T., Hoit, B. D., Liggett, S. B., Lorenz, J. N., Dorn, G. W., II, and Kranias, E. G. (2001) Circulation 103, 889 – 896 25. Mascioni, A., Karim, C., Barany, G., Thomas, D. D., and Veglia, G. (2002) Biochemistry 41, 475– 482