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.

Figure 1. Phosphorylation of PLN on serine-16 and threonine-17 and SERCA2a expression in the heart of Epac1 KO at baseline.

(A) Phosphorylation on serine-16 (Ser16) was significantly decreased in Epac1 KO compared with WT (WT versus Epac1 KO: 100% ± 6.5% versus 57% ± 7.1%, n = 6, *P < 0.01). The ratio of phosphorylated/total protein expression of PLN in WT was taken as 100% in each determination. (B) Phosphorylation on threonine-17 (Thr17) was not different in Epac1 KO and WT (WT versus Epac1 KO: 100% ± 5.7% versus 92% ± 11.5%, n = 7–10, P = NS). The ratio of phosphorylated/total protein expression of PLN in WT was taken as 100% in each determination. (C) The expression of SERCA2a protein was not different in WT and Epac1 KO at baseline (WT versus Epac1 KO: 100% ± 11.1% versus 105% ± 3.5%, n = 6–7, P = NS). The ratio of SERCA2a protein/GAPDH in WT was taken as 100% in each determination. (D) Representative immunoblotting results of phosphorylation of PLN on serine-16 (upper) and threonine-17 (middle) and SERCA2a (lower) are shown. T-PLN, total PLN; p-PLN, phosphorylated PLN.

Figure 2. Effects of EPAC activation on PLN phosphorylation in neonatal rat cardiac myocytes.

(A and B) Treatment of neonatal rat cardiac myocytes with 8-CPT-AM (10 μM). PLN phosphorylation on serine-16 was significantly increased at 15 minutes and remained significantly (*P < 0.05 or **P < 0.01 versus 0 minutes, n = 4) greater at 120 minutes than at 0 minutes (A). PLN phosphorylation on threonine-17 was also significantly increased at 15 minutes after treatment. However, increase fell below significance at 60 minutes versus 0 minutes and remained unchanged at 120 minutes (P = NS, versus 0 minutes, n = 4) (B). Ratio of phosphorylated/total protein expression of PLN at baseline (0 min: Ctrl) was taken as 1-fold. (C) EPAC-mediated PLN phosphorylation on serine-16 was examined in neonatal rat cardiac myocytes transfected with PKCε siRNA or control siRNA (*P < 0.01, n = 5–7). Ratio of phosphorylated/total protein expression of PLN in cells transfected with control siRNA at baseline was taken as 100%. (D) PLN phosphorylation on serine-16 was examined in cells treated with Bnz-cAMP (50 μM) and/or 8-CPT-AM (10 μM). (^##P < 0.001 versus Bnz-cAMP [50 μM] alone; ^††P < 0.001 versus 8-CPT-AM [10 μM] alone). A similar tendency was observed when 10 μM Bnz-cAMP and 5 μM 8-CPT-AM were used together (^#P < 0.001 versus Bnz-cAMP [10 μM] alone; ^†P < 0.001 versus 8-CPT-AM [5 μM] alone, n = 4–8; *P < 0.01 versus baseline, n = 4–8). Ratio of phosphorylated/total protein expression of PLN at baseline was taken as 100%.

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.

(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.

Figure 4. Ca²⁺ transient of adult isolated cardiac myocytes from Epac1 KO.

(A and B) Typical recordings of Ca²⁺ transients in cardiac myocytes from WT (A) and Epac1 KO (B). Note that the basal and peak Ca²⁺ transient amplitude and decay rate are smaller in Epac1 KO (n = 32 cells from 4 animals each). (C and D) Ca²⁺ transient parameters of isolated cardiac myocytes from Epac1 KO and WT. The basal Ca²⁺ concentration (WT versus Epac1 KO: 139 ± 6.8 versus 99 ± 11.8 nM) and peak Ca²⁺ concentration (WT versus Epac1 KO: 883 ± 35.3 versus 559 ± 33.2 nM) were significantly lower in Epac1 KO than in WT (C). The decay time constant (τ) was significantly larger in Epac1 KO than in WT (WT versus Epac1 KO: 108 ± 6.6 versus 187 ± 13.2 ms) (n = 32 cells, *P < 0.05) (D). (E and F) Typical recordings of Ca²⁺ transients after caffeine (10 mM) treatment of cardiac myocytes from WT (E) and Epac1 KO (F). (G) Peak Ca²⁺ concentration after caffeine (10 mM) treatment was significantly decreased in Epac1 KO compared with WT (WT versus Epac1 KO: 1210 ± 143 versus 835 ± 87.6 nM, n = 7, *P < 0.05).

Figure 5. Comparison of cardiac hypertrophy after aortic banding (TAC) in WT and Epac1 KO.

(A) Pressure gradients were not different in WT and Epac1 KO at 3 weeks after TAC (n = 13-16, P = NS). (B and C) LV weight (mg)/tibial length (mm) ratio (B) and LV weight (mg)/body weight (BW, g) ratio (C) were determined at 3 weeks. The degree of cardiac hypertrophy was increased at 3 weeks, but was similar in WT and Epac1 KO (LV/tibial length ratio for WT versus Epac1 KO: 7.3 ± 0.2 versus 7.0 ± 0.2; LV/BW ratio for WT versus Epac1 KO: 4.4 ± 0.1 versus 4.4 ± 0.2, n = 18–20, P = NS). (D and E) Representative gross LV section of Masson-trichrome staining in sham-operated and TAC-operated WT and Epac1 KO heart. Note that the degree of cardiac hypertrophy was similar, but the fibrotic area after aortic banding was smaller in Epac1 KO than that in WT.

Figure 6. Changes in LV function at 3 weeks after aortic banding (TAC) in WT and Epac1 KO.

Echocardiographic measurements of LV function were performed at 3 weeks after TAC of WT and Epac1 KO and in sham-operated controls. (A and B) LVEF was significantly decreased in WT (*P < 0.01), but not in Epac1 KO (P = NS) at 3 weeks (WT versus Epac1 KO: from 70% ± 0.8% to 54% ± 2.0% versus from 60% ± 1.1% to 59% ± 0.7%, n = 17–30) (A). The data were compared with those from sham-operated controls at 3 weeks in each mouse. Change of LVEF from sham-operated controls at 3 weeks after TAC was significantly greater in WT than that in Epac1 KO (B) (n = 17–31, *P < 0.01). (C and D) LVESD was significantly increased in WT, but not in Epac1 KO at 3 weeks after TAC (C). The data were compared with those from sham-operated controls at 3 weeks. Change of LVESD from sham-operated controls at 3 weeks after banding was greater in WT than that in Epac1 KO (n = 17–31, *P < 0.01) (D). The decrease of LVESD in response to intravenous acute ISO infusion (0, 0.13, 0.27, 0.40 mg/kg/min for 5 minutes) was depressed in WT, but not in Epac1 KO (n = 4–5, *P < 0.01). (E and F) The increase of LVEF (E) and the decrease of LVESD (F) in response to intravenous acute ISO infusion (0, 0.13, 0.27, 0.40 mg/kg/min for 5 minutes) were depressed in WT, but not in Epac1 KO (n = 4–5, *P < 0.01) (E).

Figure 7. Accelerated morphological deterioration after aortic banding (TAC) was attenuated in Epac1 KO.

(A) Representative images of Masson-trichrome–stained sections of sham-operated and TAC-operated heart of WT and Epac1 KO at 3 weeks. Scale bars: 100 μm. (B) Quantitative analysis of the fibrotic area in sham-operated and TAC-operated heart at 3 weeks after TAC of WT and Epac1 KO. Cardiac fibrosis was significantly increased after TAC in WT and Epac1 KO, but magnitude of the increase was much smaller in Epac1 KO (n = 4, *P < 0.01). (C) TUNEL-positive myocytes in LV myocardium were counted in WT and Epac1 KO and expressed as percentage of total myocytes. Number of TUNEL-positive myocytes was significantly smaller in Epac1 KO than in WT at 3 weeks after aortic banding (n = 4–6, *P < 0.05). (D) Expression of BAX protein was compared between WT and Epac1 KO. Protein expression of BAX was determined by Western blot analysis, which showed greater expression in WT than in Epac1 KO at 3 weeks after TAC (n = 4, *P < 0.05). Expression of BAX protein in the heart of sham-operated control WT was taken as 100%. Representative immunoblotting results are shown. (E) Representative images of double-immunostaining for dystrophin (brown) and PECAM (blue) of WT and Epac1 KO at baseline and 3 weeks after aortic banding. Scale bars: 50 μm. (F) Number of microvessels per cardiomyocyte was compared in WT and Epac1 KO at baseline and at 3 weeks after aortic banding (n = 4, *P < 0.01).

Figure 8. The duration of AF induced by transesophageal rapid atrial pacing was decreased in Epac1 KO.

(A and B) Transesophageal pacing was performed at a cycle length of 30 ms for 1 minute, and the duration of pacing-induced AF was examined in Epac1 KO and WT (A). The duration of AF was significantly decreased in Epac1 KO compared with WT controls. AF was induced in all WT mice and its duration was 59 ± 3.4 seconds (n = 4). In Epac1 KO, we found that AF was hardly inducible (1.4 ± 1.0 seconds, n = 5, **P < 0.01 versus WT) (B).

Figure 9. A schematic model of cAMP/EPAC signaling as opposed to cAMP/PKA signaling in the heart.

Ca²⁺ stored in the SR is released into the cytosol to activate cardiac muscle contraction and subsequently reaccumulated to promote relaxation. PLN phosphorylation on serine-16 as well as threonine-17 occurs via the EPAC/PLC/PKCε/CaMKII pathway (27, 28). However, under physiological conditions, PLN phosphorylation on serine-16 by PKA rather than on threonine-17 by CaMKII is the major regulator of Ca²⁺ cycling in the heart (56, 57). Our current study indicates that PLN on serine-16 and RyR2 on serine-2808 and serine-2814 are phosphorylated by EPAC1 in addition to and independently of PKA or CaMKII. More importantly, hyperphosphorylation of PLN on serine-16 was recently reported to be associated not only with an increase in cardiac function in young animals (16, 17), but also with arrhythmia and cardiomyopathy after adrenergic stress, aortic banding, or ischemia (18, 20, 21). Our results suggest that Epac1-mediated hyperphosphorylation of PLN and RyR2 might be required for the development of heart failure as well as arrhythmia, in addition to PKA- or CaMKII mediated activation.