Epac1-dependent phospholamban phosphorylation mediates the cardiac response to stresses
Satoshi Okumura, Takayuki Fujita, Wenqian Cai, Meihua Jin, Iyuki Namekata, Yasumasa Mototani, Huiling Jin, Yoshiki Ohnuki, Yayoi Tsuneoka, Reiko Kurotani, Kenji Suita, Yuko Kawakami, Shogo Hamaguchi, Takaya Abe, Hiroshi Kiyonari, Takashi Tsunematsu, Yunzhe Bai, Sayaka Suzuki, Yuko Hidaka, Masanari Umemura, Yasuhiro Ichikawa, Utako Yokoyama, Motohiko Sato, Fumio Ishikawa, Hiroko Izumi-Nakaseko, Satomi Adachi-Akahane, Hikaru Tanaka, Yoshihiro Ishikawa
Satoshi Okumura, Takayuki Fujita, Wenqian Cai, Meihua Jin, Iyuki Namekata, Yasumasa Mototani, Huiling Jin, Yoshiki Ohnuki, Yayoi Tsuneoka, Reiko Kurotani, Kenji Suita, Yuko Kawakami, Shogo Hamaguchi, Takaya Abe, Hiroshi Kiyonari, Takashi Tsunematsu, Yunzhe Bai, Sayaka Suzuki, Yuko Hidaka, Masanari Umemura, Yasuhiro Ichikawa, Utako Yokoyama, Motohiko Sato, Fumio Ishikawa, Hiroko Izumi-Nakaseko, Satomi Adachi-Akahane, Hikaru Tanaka, Yoshihiro Ishikawa
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Research Article Cardiology

Epac1-dependent phospholamban phosphorylation mediates the cardiac response to stresses

Abstract

PKA phosphorylates multiple molecules involved in calcium (Ca2+) handling in cardiac myocytes and is considered to be the predominant regulator of β-adrenergic receptor–mediated enhancement of cardiac contractility; however, recent identification of exchange protein activated by cAMP (EPAC), which is independently activated by cAMP, has challenged this paradigm. Mice lacking Epac1 (Epac1 KO) exhibited decreased cardiac contractility with reduced phospholamban (PLN) phosphorylation at serine-16, the major PKA-mediated phosphorylation site. In Epac1 KO mice, intracellular Ca2+ storage and the magnitude of Ca2+ movement were decreased; however, PKA expression remained unchanged, and activation of PKA with isoproterenol improved cardiac contractility. In contrast, direct activation of EPAC in cardiomyocytes led to increased PLN phosphorylation at serine-16, which was dependent on PLC and PKCε. Importantly, Epac1 deletion protected the heart from various stresses, while Epac2 deletion was not protective. Compared with WT mice, aortic banding induced a similar degree of cardiac hypertrophy in Epac1 KO; however, lack of Epac1 prevented subsequent cardiac dysfunction as a result of decreased cardiac myocyte apoptosis and fibrosis. Similarly, Epac1 KO animals showed resistance to isoproterenol- and aging-induced cardiomyopathy and attenuation of arrhythmogenic activity. These data support Epac1 as an important regulator of PKA-independent PLN phosphorylation and indicate that Epac1 regulates cardiac responsiveness to various stresses.

Authors

Satoshi Okumura, Takayuki Fujita, Wenqian Cai, Meihua Jin, Iyuki Namekata, Yasumasa Mototani, Huiling Jin, Yoshiki Ohnuki, Yayoi Tsuneoka, Reiko Kurotani, Kenji Suita, Yuko Kawakami, Shogo Hamaguchi, Takaya Abe, Hiroshi Kiyonari, Takashi Tsunematsu, Yunzhe Bai, Sayaka Suzuki, Yuko Hidaka, Masanari Umemura, Yasuhiro Ichikawa, Utako Yokoyama, Motohiko Sato, Fumio Ishikawa, Hiroko Izumi-Nakaseko, Satomi Adachi-Akahane, Hikaru Tanaka, Yoshihiro Ishikawa

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Figure 3

PLN phosphorylation on serine-16 and threonine-17 and CaMKII phosphorylation on threonine-286 in isolated WT or Epac1 KO heart perfused according to Langendorf method with or without subsequent ISO (0.1 μM) for 5 minutes.

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PLN phosphorylation on serine-16 and threonine-17 and CaMKII phosphoryla...
(A) PLN phosphorylation on serine-16 was significantly increased (*P < 0.05, **P < 0.01) in response to ISO in WT and Epac1 KO (WT: from 100% ± 7.0% to 351% ± 32%, n = 6–8; Epac1 KO: from 60 ± 2.6 to 153% ± 9%, n = 5), but increase was significantly smaller in Epac1 KO (153% ± 9%) compared with WT (351% ± 32%, **P < 0.01, n = 5–6). Ratio of phosphorylated/total protein expression of PLN in WT at baseline was taken as 100%. (B) PLN phosphorylation on threonine-17 was similar in WT and Epac1 KO at baseline and was significantly increased in response to ISO in WT (from 100% ± 2.3% to 183% ± 14%, **P < 0.01, n = 6–8) and Epac1 KO (from 94% ± 6.7% to 173% ± 13%, **P < 0.01, n = 6–8). Magnitudes of the increase were similar (P = NS). Ratio of phosphorylated/total protein expression of PLN in WT at baseline was taken as 100%. (C) CaMKII phosphorylation on threonine-286 was similar in WT and Epac1 KO at baseline and was significantly increased (**P < 0.01) in response to ISO in WT (from 100% ± 12% to 297% ± 37%, n = 5) and Epac1 KO (from 82% ± 7.7% to 278% ± 58%, n = 5). Magnitudes of increase were similar (P = NS). Ratio of phosphorylated/total protein expression of CaMKII in WT at baseline was taken as 100%. (D) Representative immunoblotting results of phosphorylation of PLN on serine-16 (upper) and threonine-17 (middle) and CaMKII on threonine-286 (lower). p-CaMKII, phosphorylated CaMKII; T-CaMKII, total CaMKII.