Biochemical and Biophysical Research Communications 325 (2004)

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1 Biochemical and Biophysical Research Communications 325 (2004) BBRC Junctophilin type 2 is associated with caveolin-3 and is down-regulated in the hypertrophic and dilated cardiomyopathies Susumu Minamisawa a,f, *,1, Jin Oshikawa a,1, Hiroshi Takeshima b, Masahiko Hoshijima c, Yibin Wang d, Kenneth R. Chien c, Yoshihiro Ishikawa a,e, Rumiko Matsuoka f,g, * a Department of Physiology, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama , Japan b Department of Medical Chemistry, Graduate School of Medicine, Tohoku University, Japan c The Institute of Molecular Medicine, University of California at San Diego, USA d Departments of Anesthesiology and Medicine, David Geffen School of Medicine, University of California at Los Angeles, USA e Departments of Medicine (Cardiology) and Cell Biology and Molecular Medicine, Cardiovascular Research Institute, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, USA f Department of Pediatric Cardiology, Tokyo WomenÕs Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo , Japan g Division of Genomic Medicine, Institute of Advanced Biomedical Engineering and Science, Graduate School of Medicine, Tokyo Women s Medical University, Tokyo , Japan Received 18 October 2004 Available online 6 November 2004 Abstract Functional coupling between the sarcolemmal membrane and the sarcoplasmic reticulum is based on distinct structures called junctional membrane complexes (JMCs). Recently, junctophilins are found to be responsible for normal formation of JMCs. In the present study, we found that junctophilin type 2 (JP-2), a unique isoform in the heart, was localized in caveolin-rich membranes, and that the expression of JP-2 was up-regulated during normal development and down-regulated in a hypertrophic or a dilated cardiomyopathic mouse model. The expression levels of JP-2 may be associated with the development of T-tubules and impaired Ca 2+ -induced Ca 2+ release in the heart. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Excitation contraction; Calcium; T-tubules; Caveolae; Cardiomyopathy; Junctional membrane complexes; Development; Hypertrophy; Gene expression All excitable cell types have a common feature of junctional membrane complexes (JMCs) between the plasma membrane and endoplasmic/sarcoplasmic reticulum (ER/SR), which mediate crosstalk between cell surface and intracellular ion channels. The crosstalk between L-type Ca 2+ channels (the dihydropyridine recep- * Corresponding authors. Fax: (S. Minamisawa), (R. Matsuoka). addresses: sminamis@med.yokohama-cu.ac.jp (S. Minamisawa), rumiko@imcir.twmu.ac.jp (R. Matsuoka). 1 These authors contributed equally to this work. tor: DHPR) on the sarcolemmal membrane and the ryanodine receptor (RyR) on the SR is a fundamental feature of excitation contraction (EC) coupling in the heart. Sarcolemmal membrane Ca 2+ current (I Ca ) triggers a subsequent larger Ca 2+ release from the SR through the activated RyR. This process is known as Ca 2+ -induced Ca 2+ release (CICR). Morphologically, the junctional SR closely faces the sarcolemmal membrane and its specific invaginations, the transverse tubules (T-tubules) in the heart. The JMCs are called peripheral coupling at the sarcolemmal membrane or diad at T-tubules. Large population of the proteins X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi: /j.bbrc

2 S. Minamisawa et al. / Biochemical and Biophysical Research Communications 325 (2004) involved in CICR is concentrated at the diad in the adult mammalian ventricles [1]. The JMCs are highly conserved structures, and the gap size between the sarcolemmal membrane and the SR is always 12 nm in matured cardiomyocytes [2,3]. Therefore, the conserved structure of the JMCs itself, especially at T-tubules, may play a fundamental role in maintaining normal functional coupling of CICR. Junctophilins (JPs) have been recently identified as a membrane spanning protein that contributes to the formation of the JMCs in excitable cells. Accumulating evidence suggests that JP is an essential component for the stabilization of the JMCs [3 5]. Four isoforms of JP have been identified so far, namely JP type 1, 2, 3, and 4 [6]. Skeletal muscles express both JP type 1 (JP-1) and type 2 (JP-2), while cardiac muscles express only JP-2. JP type 3 (JP-3) is expressed exclusively in the brain. JP anchors the ER/SR to the plasma membrane. The genetic ablation of JP-2 in mice caused wider gap size of the JMCs and deficient [Ca 2+ ] i transients in cardiomyocytes, resulting in embryonic lethality [3]. These findings indicate that JP-2 is essential to form normal JMCs and efficient CICR in the heart. Similarly, JP-1 knockout mice died shortly after birth and exhibited deficiency of triad junctions and contraction in skeletal muscles [4]. Mutations in the JP-3 gene have been reported to be associated with HuntingtonÕs diseaselike-2 in human, in which patients display characteristic motor disorders [7]. JP-3 knockout mice displayed motor discoordination [8]. These results suggest that the disruption of JPs impairs the formation of JMCs and the coupling between the plasma membrane and the ER/SR in striated muscle and neuron. A decrease in [Ca 2+ ] i transient is a common feature in cardiac myocytes from cardiomyopathic or failing hearts, while I Ca often remains unchanged. The loss of functional coupling of CICR leads to, at least in part, the decrease in [Ca 2+ ] i transient [9]. Changes in the structure and protein expression of T-tubules have been found during cardiac development [10,11] and in heart failure [12 15], indicating that they may play an important role in functional uncoupling of CICR in heart failure. Accordingly, alternations in the expression of JP-2 may be associated with the impaired formation of the T-tubules and/or the JMCs in cardiomyopathic hearts. In the present study, we analyzed the JP-2 expression during cardiac development and in two different animal models of cardiomyopathies. Materials and methods Animals and tissue samples. ICR mice were obtained from Clea Japan (Tokyo). Embryos from ICR mice were obtained from timedpregnant animals. The transgenic model of hypertrophic or dilated cardiomyopathy was established by ventricle-specific over-expression of activated H-ras transgene (RASOE) or ablation of muscle-specific LIM gene (MLPKO), respectively. These animal models were well characterized elsewhere [16,17]. Atrial and left ventricular myocardium were separated and stored at 80 C. Western blot and sucrose gradient analyses. The crude membrane preparations were made as previously described [18]. The 30 lg of the crude membranes was separated by SDS polyacrylamide gel electrophoresis (PAGE) and transferred to PVDF membranes. The antibody that recognizes JP-2 was prepared as previously described [3]. Monoclonal anti-caveolin 3 (cav-3) antibody (BD Bioscience, CA, USA) was purchased from manufacturers. The immunoreactive bands were visualized as previously described [19]. Immunoprecipitation assay. The heart tissues were lysed in a buffer containing 25 mm Tris HCl (ph 8.0), 10 mm EGTA, 10 mm EDTA, 10 mm Na 4 P 2 O 7, 10 mm Na 3 VO 4, 0.1 M NaF, 1% Triton X-100, 60 mm octylglucoside, and protease inhibitors. The lysates were incubated with antibodies against cav-3 for overnight. Non-immune mouse IgG was used in a control experiment. The immune complexes formed by the addition of protein G Sepharose were incubated for 2 h, followed by washing three times. Bound proteins were solubilized and analyzed on SDS PAGE, followed by immunoblotting for JP-2. Northern blot analysis. Total RNA was isolated and Northern blot analysis was performed as previously described [20]. JP-2 cdna probe was prepared as previously described [3]. Results and discussion First, we investigated the subcellular localization of JP-2 in the mouse ventricular myocytes. Using discontinuous sucrose gradient and Western blot analyses, we found that JP-2 proteins localized to low-density membrane fractions together with cav-3, a muscle-specific structural protein of caveolae (Fig. 1A). The data suggested that JP-2 is also associated with caveolae or lipid rafts. Moreover, immunoprecipitation assay revealed that JP-2 physically interacts with cav-3 (Fig. 1B). Caveolae, infoldings of lipid rafts in the plasma membrane, are also localized in T-tubules of ventricular myocytes [21]. The DHPRs are densely localized in caveolae [22,23] and in T-tubules [1,24]. A recent study demonstrated that deficiency of cav-3 resulted in diffuse Fig. 1. (A) Immunoblot analysis of samples obtained by sucrose gradient fractionation. JP-2 protein is detected in caveolin-rich fraction (fractions 5, 6, and 7), with cav-3 immunoblot shown for comparison. (B) Immunoprecipitation assay of JP-2. The extracts of mouse whole left ventricle were processed for immunoprecipitation with cav-3 antibodies, as described in Materials and methods. Non-immune mouse IgG was used in a control experiment. Bound proteins were solubilized and analyzed on SDS PAGE, followed by immunoblotting for JP-2. JP-2, junctophilin type 2; cav-3, caveolin-3.

3 854 S. Minamisawa et al. / Biochemical and Biophysical Research Communications 325 (2004) distribution of the DHPRs and the RyR in skeletal muscle [25], indicating that caveolae are required for densely coupled co-localization of the DHPRs and the RyR in the JMCs. Our data suggest that JP-2 is also co-localized with the cav-3-mediated protein complex to form normal JMCs and efficient CICR in the heart. Since there is no putative transmembrane segment in the amino-terminal end of JP [3], a further study whether JP is anchored to the plasma membrane via cav-3 would be intriguing. Next, we examined the developmental changes in the expression of JP-2 mrna and protein in the heart by Northern and Western blot analyses. JP-2 mrna and protein expression were already detectable in the ventricles at embryonic day 12.5 and increased with development (Figs. 2A and B). The levels of JP-2 mrna in the atria were approximately 60% of those in the ventricles and were also increased with development (Fig. 2C). Embryonic and neonatal cardiac myocytes exhibit absent or very poor T-tubules, which develop within the first few weeks of life. Even in the adult atrial myocytes, T-tubules are absent or far less developed [1]. The lower level of JP-2 may reflect the sparse T-tubule system during early stages of development and in the atria. The present result suggested that the increased expression of JP-2 is associated with development of the JMCs, especially diad, and contribute to effective functional coupling between the DHPR and the RyR in mature cardiomyocytes. Neither JP-1 nor JP-3 mrna was Fig. 2. The expression of JP-2 during cardiac development. (A) A representative result of JP-2 mrna expression during cardiac development. The left ventricle tissues from embryonic day 12.5 (ed12.5) to postnatal one year mice were subjected to Northern blot analysis. The expression of JP-2 mrna was increased during development. (B) The expression of JP-2 protein during cardiac development was analyzed by Western blot analysis. JP-2 protein expression was increased in a similar manner to its mrna expression. (C) The expression of JP-2 mrna in the atria was analyzed by Northern blot. JP-2 mrna expression in the atria was also increased developmentally. V, ventricles; A, atria. detected in the atria and ventricles at any stages of development (data not shown), indicating that JP-2 is a unique isoform in the heart. Interestingly, a previous study demonstrated that the expression of JP-2 was not detectable in skeletal muscles until embryonic day 17 [4], suggesting that JP-2 is required for the development of JMCs earlier in the heart than in skeletal muscles. Finally, we examined the changes in the expression of JP-2 mrna and protein in the cardiomyopathic heart by Northern and Western blot analyses, because impaired EC coupling defect is a common feature in cardiomyopathy. We used the well-characterized animal models of cardiomyopathy, RASOE [16] and MLPKO [17,26,27] mice. Although RASOE and MLPKO mice displayed distinct morphological phenotypes in the ventricles, both animal models displayed depressed [Ca 2+ ] i transient with normal I Ca [16,17,26]. The suppressed SR Ca 2+ uptake plays an important role in the decreased [Ca 2+ ] i transient in RASOE mice [16] and in MLPKO mice [26]. However, impaired functional coupling of CICR may also contribute to, at least in part, the depressed [Ca 2+ ] i transient in both animal models of cardiomyopathies. Fig. 3A shows that the levels of JP-2 mrna were down-regulated by 60% in RASOE mice compared with those in non-transgenic mice. The levels of ANF mrna were 17 times higher in RASOE mice. The JP-2 protein expression was also decreased by approximately 40% in RASOE mice (Fig. 3B), indicating the simultaneous down-regulation of both JP-2 mrna and protein in RASOE mice. Fig. 4 shows the expression of JP-2 mrna was similar between wildtype and MLPKO mice while JP-2 protein expression was decreased by approximately 40% in MLPKO mice (Fig. 4B). Thus, the expression of JP-2 may be regulated posttranscriptionally in MLPKO mice. No JP-1 transcript was detected in RASOE (Fig. 3A) and MLPKO mice (data not shown), suggesting the absence of compensatory up-regulation of JP-1 in these mice. To our knowledge, the present study provided the first evidence that the expression of JP-2 is decreased in pathological hearts. Several studies have demonstrated that the density of the T-tubules is decreased in myocytes from the heart with heart failure and the structure of the T-tubules is altered [12 15]. The decrease in the expression of JP-2 may reflect the reduced density of the T-tubules in cardiomyopathy. Another possibility is that the structure of the JMC itself is altered in cardiomyopathy, although it has not been investigated in pathological hearts so far. Since the wider gap size (30 nm) in peripheral coupling is observed at small percentage of the JMCs in embryonic myocytes and disappears in adult myocytes [3], it is interesting to investigate whether this embryonic phenotype of the JMCs is induced in the course of embryonic program that is often found in cardiac hypertrophy and/or heart failure. Structural

4 S. Minamisawa et al. / Biochemical and Biophysical Research Communications 325 (2004) Fig. 3. The expression of JP2 in RASOE mice. (A) A representative result of JP-2 mrna expression in RASOE mice. The left ventricle tissues from RASOE (n = 11) and the littermate non-transgenic (NTG, n = 8) mice were subjected to Northern blot analysis. Ten micrograms of total RNA was used. The expression of JP-2 mrna was down-regulated by 60% in RASOE mice relative to that in non-transgenic mice. (B) A representative result of JP-2 protein expression in RASOE mice. The level of JP-2 protein expression was significantly decreased in RASOE mice (n = 4) compared with NTG mice (n = 4). Fig. 4. The expression of JP-2 in MLPKO mice. (A) A representative result of JP-2 mrna expression in MLPKO mice. The left ventricle tissues from MLPKO mice (n = 4) and the littermate wild-type (WT, n = 4) were subjected to Northern blot analysis. The levels of JP-2 mrna expression were comparable between MLPKO and WT mice. (B) A representative result of JP-2 protein expression in MLPKO mice. The level of JP-2 protein was significantly decreased in MLPKO mice (n = 4) when compared with WT mice (n = 4). changes in the JMCs may result in impaired CICR and the reduced expression of JP-2. In conclusion, the expression of junctophilin type 2 was up-regulated during development and was downregulated at the protein level in two distinct models of cardiomyopathies. The expression levels of JP-2 may associate with normal development of JMCs and impaired CICR in the heart.

5 856 S. Minamisawa et al. / Biochemical and Biophysical Research Communications 325 (2004) Acknowledgments We thank Hideaki Hori for professional editing of the manuscript. This work was supported in part by the Ministry of Education, Science, Sports and Culture of Japan (to S. Minamisawa, Y. Ishikawa) and a Grant for Promotion of the Advancement of Education and Research in Graduate schools (2000, 2001, 2002; to R. Matsuoka). References [1] F. Brette, C. Orchard, T-tubule function in mammalian cardiac myocytes, Circ. Res. 92 (2003) [2] D.W. Fawcett, N.S. McNutt, The ultrastructure of the cat myocardium. I. Ventricular papillary muscle, J. Cell Biol. 42 (1969) [3] H. Takeshima, S. Komazaki, M. Nishi, M. Iino, K. Kangawa, Junctophilins: a novel family of junctional membrane complex proteins, Mol. Cell 6 (2000) [4] K. Ito, S. Komazaki, K. Sasamoto, M. Yoshida, M. Nishi, K. Kitamura, H. 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