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Presenilin 2 regulates the systolic function of heart by modulating Ca²⁺ signaling

Toshihiro Takeda^*, Michio Asahi, Osamu Yamaguchi^*, Shungo Hikoso^*, Hiroyuki Nakayama^*, Yoichiro Kusakari, Makoto Kawai, Kenichi Hongo, Yoshiharu Higuchi^*, Kazunori Kashiwase^*, Tetsuya Watanabe^*, Masayuki Taniike^*, Atsuko Nakai^*, Kazuhiko Nishida^*, Satoshi Kurihara, Dorit B. Donoviel, Alan Bernstein, Taisuke Tomita^¶, Takeshi Iwatsubo^¶, Masatsugu Hori^* and Kinya Otsu^*,1

^* Department of Cardiovascular Medicine and
Department of Biochemistry, Osaka University Graduate School of Medicine, Suita, Osaka;
Department of Physiology (II), The Jikei University School of Medicine, Tokyo, Japan;
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada; and
^¶ Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan

¹Correspondence: Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: kotsu{at}medone.med.osaka-u.ac.jp

SPECIFIC AIMS

Mutations in presenilin 2 (PS2) genes account for early-onset Alzheimer’s disease, and PS2 is ubiquitously expressed in various tissues including hearts. In this study, we examined cardiac phenotypes of PS2 knockout (PS2KO) mice in order to elucidate a role of PS2 in hearts.

PRINCIPAL FINDINGS

1. Physiological characteristics of PS2KO hearts
The PS2KO hearts showed no evidence of any cardiac morphologic defects, nor did histological examination of the hearts demonstrate any myofibrillar disarray, necrosis, or ventricular fibrosis. There was no significant difference in the myocyte cross-sectional area of PS2KO and wild-type littermate (WT) mice (201.1±8.6 µm² for WT and 200.5±3.5 µm² for PS2KO). Nor were there any differences in the ratios of heart weight to body weight (4.09±0.07 mg/g for WT and 4.18±0.04 mg/g for PS2KO) and left ventricular (LV) weight to body weight (2.97±0.07 mg/g for WT and 3.11±0.04 mg/g for PS2KO) between PS2KO and WT hearts. Hemodynamic data did not indicate any differences in heart rate (478±17 bpm for WT and 463±18 bpm for PS2KO), left ventricular (LV) systolic pressure (82.0±1.3 mmHg for WT and 90.7±4.2 mmHg for PS2KO), LV end-diastolic pressure (0.86±0.36 mmHg for WT and 0.71±0.42 mmHg for PS2KO), and the minimum first derivative of the LV pressure (–dp/dt) (–5243±295 mmHg for WT and –6167±260 mmHg for PS2KO) between PS2KO and WT mice. However, the first derivative of the maximum LV pressure (+dp/dt) was significantly higher in PS2KO than in WT mice (7143±311 mmHg/s for WT and 8629±358 mmHg/s for PS2KO), suggesting that cardiac contractility significantly increased in PS2KO mice.

2. Measurement of Ca²⁺ transients and tension in left ventricular papillary muscles
Intracellular Ca²⁺ kinetics and contractile properties were measured in aequorin-loaded papillary muscle at 2 mM [Ca²⁺]_o at a stimulation frequency of 0.2 Hz (30°C) (Fig. 1 A). The length and width of the papillary muscle preparations from PS2KO and WT mice were not significantly different (Fig. 1B ). Isolated papillary muscle from PS2KO hearts exhibited a significant increase in peak amplitude of Ca²⁺ transient compared with that from WT hearts. Isometric tension measurements performed on papillary muscles from PS2KO mice indicated an increase in peak tension per cross-sectional area compared with that from WT mice. The times to peak of Ca²⁺ transient and to peak isometric tension in PS2KO and WT mice were not noticeably different, nor were the decay time of Ca²⁺ transient and the relaxation time of isometric tension. PS2KO mouse hearts exhibited no change in expression of calcium regulatory proteins (Fig. 1C ). The Ca²⁺ content of the sarcoplasmic reticulum, estimated by releasing Ca²⁺ with the addition of 50 mM caffeine showed no significant difference between PS2KO and WT (Fig. 1D ). These data suggest that the enhanced cardiac contractility shown in PS2KO hearts result in an acceleration of Ca²⁺ release from the sarcoplasmic reticulum.

View larger version (34K): [in this window] [in a new window]   Figure 1. Ca2+ transients and tension in left ventricular papillary muscles. A, B) Dissected papillary muscles (n=8 for WT and 10 for PS2KO) were microinjected with aequorin. Aequorin light signals and tensions (stimulation frequency, 0.2 Hz) were recorded simultaneously at 30°C. TPL (time-to-peak light) and TPT (time-to-peak tension), are the times measured from the onset of stimulus to the respective peak values of light and tension; decay time (the time for the light signal to decay from 75% to 25% of the peak value); relaxation time (the time required for the tension to decay from 75% to 25% of the peak value). Length and width are those of the isolated papillary muscle preparation. *P < 0.05 vs. WT. C) Western blot analysis of PS2, sorcin, and Ca2+ signaling proteins. Western blot shows C-terminal fragments of presenilin2 (PS2-CTF), ryanodine receptor2 (RyR2), sorcin (SOR), sarcoplasmic reticulum ATPase2a (SERCA2a), phospholamban (PLN), and Na-Ca exchanger (NCX) proteins detected in heart tissue from PS2KO and WT littermate mice. D, Ca2+ content of the sarcoplasmic reticulum. The saponin-treated papillary muscle or trabeculae (diameter: 150–250 µm, length: 2–5 mm) was inserted into a glass capillary tube. Ca2+ was loaded into the sarcoplasmic reticulum by activating SERCA in the presence of ATP and various concentrations of Ca2+. The amount of the released Ca2+ by the addition of 50 mM caffeine was measured with the fluorescent indicator fluo-3.

3. PS2 interacts with RyR2 and sorcin in vitro and in vivo
An earlier study demonstrated that PS2 in brain interacts with sorcin, which serves as a modulator of cardiac ryanodine receptor (RyR2), so we tested whether PS2 also interacts with RyR2. Immmunoprecipitation analysis showed that PS2, sorcin, and RyR2 interact with each other in HEK-293 cells overexpressing these proteins or in mouse hearts and brain. Immunohistochemistry of heart muscle indicated that PS2 colocalizes with RyR2 and sorcin at the Z-lines. These results indicate that PS2, RyR2, and sorcin colocalize and interact with each other in vivo as well as in vitro.

4. Effects of Ca²⁺ concentration on physical and functional interaction among PS2, RyR2, and sorcin
An increase in intracellular Ca²⁺ concentration by the treatment with Ca²⁺ ionophore, A23187, was found to enhance the sorcin/PS2 interaction. So we tested the effects of Ca²⁺ concentration on interaction among PS2, RyR2 and sorcin. Elevation of Ca²⁺ from pCa 7 to 5 resulted in a concentration-dependent decrease in the amount of PS2, which was immunoprecipitated with an antibody against RyR2, but in an increase in the amount of sorcin associated with RyR2 (Fig. 2 A). PS2/sorcin interaction was enhanced by elevation of Ca²⁺ concentration as described before. Finally, we examined the effect of extracellular Ca²⁺ concentration on the Ca²⁺ transient and tension (Fig. 2B ). We found significant increases in peak Ca²⁺ transient and tension at 1 and 2 mM of [Ca²⁺]_o in PS2KO compared with those in WT mice, but not at 4 mM [Ca²⁺]_o.

View larger version (29K): [in this window] [in a new window]   Figure 2. A) Effects of intracellular Ca2+ concentration on the physical interaction of presenilin2 with cardiac ryanodine receptor or sorcin. Calcium was added to immunoprecipitation buffer to yield free Ca2+ concentration in the range of 0.1 to 10 µM (pCa 7 to 5) and immunoprecipitation with anti-RyR2 or anti-PS2 antibodies was performed in HEK-293 cell homogenates. PS2 and sorcin (SOR) antibodies were used for immunoblotting. B) The effect of extracellular Ca2+ concentration on the Ca2+ transients and tension (n=8 for WT and 10 for PS2KO). Open and closed bars represent WT and PS2KO, respectively. *P < 0.05 vs. WT. C) Relationship between peak Ca2+ and peak tension. Open and closed marks represent WT and PS2KO, respectively (circles, [Ca2+]o=4 mM; squares, [Ca2+]o=2 mM; triangles, [Ca2+]o=1 mM).

CONCLUSIONS AND SIGNIFICANCE

In this study, we demonstrated the interaction between PS2, RyR2, and sorcin. PS2 inhibits cardiac contractility by reducing the Ca²⁺ transients. PS2 dysfunction may lead to the abnormality of Ca²⁺ homeostasis and disturb the cardiac function and be involved in the pathogenesis of heart failure.

View larger version (24K): [in this window] [in a new window]   Figure 3. Regulation of excitation-contraction coupling by PS2. PS2 and sorcin prevent release of Ca2+ by inhibiting RyR2 activity. At low Ca2+ concentration PS2 interacts with RyR2, whereas sorcin dissociates from PS2 and RyR2. In the absence of PS2, the amount of Ca2+ released from sarcoplasmic reticulum were increased compared with that in WT hearts, leading to enhanced cardiac contractility. At a high Ca2+ concentration, sorcin translocates to the membrane and binds to PS2 and RyR2, and PS2 dissociates from RyR2. As a result, the amount of Ca2+ release from sarcoplasmic reticulum in PS2 KO hearts is equal to that in WT hearts.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3744fje;

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