Epac1 increases myosin regulatory light-chain phosphorylation, energetic cost of contraction, and susceptibility to heart failure

Figures

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  Fig 1. Epac1 expression in the hearts of NTG and Epac1TG.

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  Fig 2. Phosphorylation status of myofilament proteins prepared from Epac1TG. (A) Representative SDS-PAGE patterns (10-20% gradient gel) of skinned myocardium prepared from NTG (left lane) and Epac1TG (right lane). The gel was stained with Pro-Q Diamond (specific for phosphorylated proteins) and subsequently stained with SYPRO Ruby (for total proteins). RLC, myosin regulatory light chain; TnI, troponin I; TnT, troponin T; MyBP-C, myosin binding protein-C. (B) RLC phosphorylation in Epac1TG was significantly greater than that in NTG (*P < 0.05 by unpaired t-test). However, phosphorylation levels of TnI, TnT and MyBP-C were similar between the two groups (P = NS by unpaired t-test). The mean phosphorylation level in NTG was taken as 100% in each determination. Bar blots represent means ± SD and open circles show individual data from biological replicates of NTG and Epac1TG (n = 4 each), each with 3 technical replicates.

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  Fig 3. Epac activation with 8CPT-AM induced RLC phosphorylation in the perfused hearts. (A) Representative phosphorylation status of cardiac myofibril components, RLC, TnI, TnT and MyBP-C, as determined by SDS-PAGE analysis of WT hearts perfused with or without Epac activator, 8CPT-AM (2 μM), or 8CPT-AM (2 μM) plus PKA inhibitor, H-89 (5 μM), for 30 min. The gel was stained first with Pro-Q Diamond and then stained with SYPRO Ruby. RLC, myosin regulatory light chain; TnI, troponin I; TnT, troponin T; MyBP-C, myosin binding protein-C. (B) RLC phosphorylation was significantly increased in the presence of 8CPT-AM (2 μM) (*P < 0.05 vs. Control by one-way ANOVA, n = 6 each) or 8CPT-AM (2 μM) plus H-89 (5 μm) (*P < 0.05 vs. Control by one-way ANOVA, n = 6 each), but the magnitudes of the increase were similar (P = NS by one-way ANOVA, n = 6 each). Phosphorylation levels of TnI, TnT and MyBP-C were similar among the three groups (P = NS by one-way ANOVA, n = 6 each). The mean phosphorylation level in the control was taken as 100% in each case. Bar blots represent means ± SD and open circles show individual data from biological replicates of Control, 8CPT-AM, 8CPT-AM + H89 (n = 6 each), each…

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  Fig 4. Epac activation increased RLC phosphorylation via PLC/PKC. (A) Representative SDS-PAGE patterns of skinned myocardium prepared from WT and treated with 8CPT-AM (5 μM for 30 min) in the presence or absence of PLC inhibitor (U73122, 5 μM), PKC inhibitor (BIM, 1 μM), CaMKII inhibitor (KN-93, 2 μM), MLCK inhibitor (ML-7, 10 μM) or ZIPK inhibitor (HS38, 50 μM). The gel was stained with Pro-Q Diamond and subsequently stained with SYPRO Ruby. (B) Pharmacological inhibition of CaMKII, MLCK or ZIPK did not alter the increase of RLC phosphorylation in response to 8CPT-AM (*P < 0.05, **P < 0.01 vs. Control by one-way ANOVA), but pharmacological inhibition of PLC or PKC attenuated Epac-mediated RLC phosphorylation (P = NS vs. Control by one-way ANOVA). The mean phosphorylation level in the control was taken as 100% in each determination. (C) Scheme illustration of phosphorylation and dephosphorylation of RLC. Myosin phosphatase is composed of a 37-kDa catalytic subunit (PP1cδ) that is responsible for dephosphorylation of its highly specific substrate (RLC) and 130-kDa regulatory myosin binding subunit (MYPT). Phosphorylation of MYPT by Rho kinase results in loss of myosin phosphatase…

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  Fig 5. Epac activation increased MYPT phosphorylation via PLC/PKC. (A) Representative western blotting of phosphorylated (Thr 696) and total MYPT1 as well as phosphorylated (Thr 646) and total MYPT2 in skinned myocardium prepared from NTG and Epac1TG and treated with or without 8CPT-AM (1 μM or 5 μM for 30 min). (B, C) In NTG, phosphorylation of MYPT1 (Thr 696) (B; left) and MYPT2 (Thr 646) (C; left) were not altered by the 8CPT-AM treatment at 1 μM (P = NS vs. Control (NTG) by one-way ANOVA), but those were significantly increased by the 8CPT-AM treatment at 5 μM (*P < 0.05 vs. Control (NTG) by one-way ANOVA). In Epac1TG, phosphorylation of MYPT1 (B; right) and MYPT2 (C; right) might be saturated without 8CPT-AM and after 8CPT-AM treatment (1 μM and 5 μM) (*P < 0.05, **P < 0.01 vs. Control (NTG) by one-way ANOVA). The mean phosphorylation level in the control NTG was taken as 100% in each determination. (D) Representative western blotting of phosphorylated and total MYPT1/2 in skinned myocardium treated with 8CPT-AM (5 μM for 30 min) in the presence or absence of PLC inhibitor (U73122, 5 μM), PKC inhibitor (BIM, 1 μM), CaMKII inhibitor (KN-93, 2 μM), MLCK inhibitor (ML-7, 10 μM) or…

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  Fig 6. Functional characterization of skinned myocardium prepared from Epac1TG. (A, B) Average values of isometric force (A) and ATPase activity (B) at pCa 6.1, 5.8, 5.5, 5.1 and 4.6 were plotted for NTG (open circle) and Epac1TG (closed circle), and the data points were fitted to the Hill equation (solid line, NTG; dashed line, Epac1TG). (C) Average pCa50 values (Ca2+ concentration required for half maximal effect) of isometric force (left) and ATPase activity (right) were significantly greater in Epac1TG than in NTG (*P < 0.05 by unpaired t-test). (D) Average Hill coefficient of isometric force (left) and ATPase activity (right) were similar in NTG and Epac1TG (P = NS by unpaired t-test). (E) Maximal isometric force at pCa 4.6 was similar in NTG and Epac1TG (P = NS by unpaired t-test). (F-G) Maximal ATPase activity (F) and tension cost at pCa 4.6 (G) were significantly greater in Epac1TG than in NTG (*P < 0.05 by unpaired t-test). Bar blots represent means ± SD and open circles show individual data from biological replicates of NTG and Epac1TG (n = 4 each), each with 3 technical replicates.

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  Fig 7. Effects of chronic ISO infusion on cardiac function in Epac1TG. (A) LVEF was similar between NTG and Epac1TG in the vehicle-treated group (P = NS by one-way ANOVA). LVEF in the ISO-treated group was significantly smaller than that in the vehicle-treated group in both NTG (*P < 0.05 by one-way ANOVA) and Epac1TG (**P < 0.01 by one-way ANOVA), but LVEF in ISO-treated Epac1TG was much smaller than that in ISO-treated NTG (**P < 0.01 by one-way ANOVA). (B) Change of LVEF from the vehicle- to the ISO-treated group was significantly greater in Epac1TG than in NTG (*P < 0.05 by unpaired t-test). (C) LVESD was similar between the vehicle-treated group and the ISO-treated group in NTG (P = NS by one-way ANOVA), but was significantly greater in the ISO-treated group than in the vehicle-treated group in Epac1TG (*P < 0.05 by one-way ANOVA). (D) Change of LVESD from vehicle- to ISO-treated group was significantly greater in Epac1TG than in NTG (*P < 0.05 by unpaired t-test). (E) LV mass/tibial length ratio in vehicle-treated Epac1TG was significantly greater than that in vehicle-treated NTG (**P < 0.01 by one-way ANOVA). It was also significantly greater in the ISO-treated group than in the…

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  Fig 8. Schematic summary of the proposed role of Epac1 in the regulation of myofilament function. This scheme illustrates the proposed relationship between PKA (left) and Epac1 (right) in the regulation of Ca2+ sensitivity and tension cost in cardiac myofilaments. Epac1-mediated increase of Ca2+ sensitivity and decrease of tension cost represent findings in this study (blue-shaded columns) and PKA-mediated decrease of Ca2+ sensitivity and increase of tension cost (dark-shaded columns) represent findings reported previously [24,73].