Extracellular and Intracellular Angiotensin II Regulate the Automaticity of Developing Cardiomyocytes via Different Signaling Pathways

Figures

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  FIGURE 1. Ang II, AT1R, and AT2R were detected in mESC-CMs and NRVMs. (A) Representative images showing the immunocytochemistry of iAng II in mESC-CMs. Cells were immunostained for cTnT (red), Ang II (green), and the nucleus with DAPI (blue). The results indicated that Ang II was present in mESC-CMs. (B) Linear calibration curve of Ang II. (C) Analysis of the NRVM samples by UHPLC-ESI-MS/MS. The extracted ion chromatograms of the two kinds of MRM. The upper one is the 523.8 ± 0.1 m/z →263.1 ± 0.1 m/z; the lower one is the 523.8 ± 0.1 m/z →784.4 ± 0.1 m/z. (D) mESC-CMs were transduced with the vector carrying AT1R-YFP and were stained with DAPI (blue). AT1R-YFP signals were detected on the plasma membrane. (E, F) mESC-CMs were stained with DAPI (blue), anti-AT2R (green), and (E) anti-cTnT (red) or (F) anti-SERCA2 (red). Merged images revealed extensive co-localization of AT2R with the nucleus and the SERCA2, suggesting that AT2R was located predominately on the nucleus and the SR. Scale bars represent 10 μm.

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  FIGURE 2. Application of exogenous Ang II increased APs transiently, and the effect could be attenuated by the AT1R blocker but not the AT2R blocker. (A) Ang II increased the APs of mESC-CMs in a transient manner. Raw traces of spontaneous APs at the basal level and upon treatment with 10–7 M Ang II in mESC-CMs (left panel). Summarized data on the AP rate and the DD slope of APs (right panel). (B) AT1R blocker attenuated Ang II–induced changes in APs. Raw traces of spontaneous APs at the basal level and upon treatment with 50 μM losartan, followed by the subsequent application of 10–7 M Ang II in mESC-CMs (left panel). Summarized data on the AP rate and the DD slope of APs at the basal level or upon treatment with 50 μM losartan, followed by 10–7 M Ang II (right panel). Losartan attenuated Ang II–induced changes in the parameters of APs. (C) AT2R blocker did not affect Ang II–induced changes in APs. Raw traces of spontaneous APs at the basal level and upon treatment with 10 μM PD123319, followed by the subsequent application of 10–7 M Ang II in mESC-CMs (left panel). Summarized data on the AP rate and the DD slope of APs at the basal level or upon treatment with 10 μM PD123319, followed by…

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  FIGURE 3. Dual current patch clamp is an advantageous approach to investigate the effect of intracellular delivery of drugs on the APs of mESC-CMs reliably. (A) Ang II-FITC can be successfully delivered into mESC-CMs using the glass electrode as shown by the appearance of the green fluorescence signal inside the cell. (B) Calcium indicator Fluo-4-IM can be delivered into mESC-CMs without a leak from the glass electrode. (C) Bright view of a dual current patch clamp experiment of mESC-CMs (left panel). A schematic diagram on the dual patch clamp experimental configuration (right panel). In dual patch clamp, AP measurement will first be made using the “recording electrode” [as shown by black traces in (D) and (E); on the other hand, red traces in (D) and (E) represent recording in the “drug electrode”]. Before the membrane breakage by the “drug electrode”, the recording in the “recording electrode” serves as the “basal” level recording. After membrane breakage of the “drug electrode” and thereby the delivery of the drug to the intracellular environment, the AP measured in the “recording electrode” represents the effect of the drug. (D, E) cAMP was used as a positive control to show the…

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  FIGURE 4. iAng II decreased the APs of mESC-CMs in a persistent manner. (A) Dual current patch clamp of APs of mESC-CMs without intracellular drug delivery (i.e., before membrane breakage by the “drug electrode”). Upper panel represents recording in the “drug electrode” while the lower panel represents recording in the “recording electrode.” (B) Dual current patch clamp of APs of mESC-CMs with intracellular delivery of Ang II (i.e., after membrane breakage by the “drug electrode”). Upper panel represents recording in the “drug electrode” while the lower panel represents recording in the “recording electrode.” (C) Traces labeled as pink in (A) (represents APs before iAng II delivery) and as blue in (B) (represents APs after iAng II delivery) are overlaid for comparison. (D–G) Summarized data on the (D) AP rate, (E) DD slope, (F) APD 50, and (G) MDP of APs upon treatment with iAng II. Values are mean ± SEM of 6–10 independent experiments. *p < 0.05 vs. control group.

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  FIGURE 5. Intracellular delivery of AT2R activator C21 decreased the APs of mESC-CMs. (A) Dual current patch clamp of APs of mESC-CMs without intracellular drug delivery (i.e., before membrane breakage by the “drug electrode”). Upper panel represents recording in the “drug electrode” while the lower panel represents recording in the “recording electrode.” (B) Dual current patch clamp of APs of mESC-CMs with intracellular delivery of C21 (i.e., after membrane breakage by the “drug electrode”). Upper panel represents recording in the “drug electrode” while the lower panel represents recording in the “recording electrode.” (C) Traces labeled as pink in (A) (represents APs before intracellular C21 delivery) and as blue in (B) (represents APs after intracellular intracellular C21 delivery) are overlaid for comparison. (D–G) Summarized data on the (D) AP rate, (E) DD slope, (F) APD50, and (G) MDP of APs upon treatment with intracellular C21. Values are mean ± SEM of 6–10 independent experiments. *p < 0.05 vs. control group.

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  FIGURE 6. iAng II attenuated caffeine-induced changes of APs and caffeine-induced Ca2+ release in mESC-CMs. (A) Raw traces of spontaneous APs at the basal level and upon treatment with caffeine (5 μM) in mESC-CMs. (B) Raw traces of spontaneous APs upon treatment with iAng II (10–7 M) followed by the subsequent application of caffeine in mESC-CMs. (C–F) Summarized data on the (C) AP rate, (D) DD slope, (E) APD50, and (F) MDP upon treatment with caffeine (5 μM) alone or upon treatment with iAng II (10–7 M) followed by caffeine (5 μM). (G) Raw traces of changes in Fluo-4 fluorescence at the basal level and upon treatment with caffeine (5 μM) in mESC-CMs. (H) Raw traces of changes in Fluo-4 fluorescence with caffeine in the presence of iAng II (10–7 M) in mESC-CMs. (I) Summarized data on the effects of iAng II (10–7 M) on the caffeine-induced Ca2+ release in mESC-CMs when external Ca2+ was absent. Values are mean ± SEM of 6–10 independent experiments. *p < 0.05 vs. control group.

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  FIGURE 7. Schematic representation of the different signaling pathways that extracellular Ang II and iAng II utilize to regulate the automaticity of CMs. Extracellular Ang II, upon binding to AT1R, leads to the production of DAG and IP3, which open TRPC3/C6 and subsequently LTCC and IP3R, respectively, and increases the automaticity of CMs. On the other hand, binding of iAng II to AT2R decreases the automaticity of CMs by reducing the activity of RyR2.