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Angiotensin II Type 1 Receptor-Mediated Electrical Remodeling in Mouse Cardiac Myocytes.

Kim J, Gao J, Cohen IS, Mathias RT - PLoS ONE (2015)

Bottom Line: Pre-application of the microtubule destabilizing agent colchicine eliminated A2 effects on Ito and AP duration, suggesting these effects depend on intracellular trafficking.Application of the biased agonist SII ([Sar1-Ile4-Ile8]A2), which stimulates receptor internalization without G protein activation, caused Ito reduction and AP prolongation similar to A2-induced changes.Moreover, all measured properties parallel those measured in dog heart, suggesting an autocrine RAS may be a fundamental feedback system that is present across species.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology & Biophysics, State University of New York at Stony Brook, Stony Brook, New York, United States of America.

ABSTRACT
We recently characterized an autocrine renin angiotensin system (RAS) in canine heart. Activation of Angiotensin II Type 1 Receptors (AT1Rs) induced electrical remodeling, including inhibition of the transient outward potassium current Ito, prolongation of the action potential (AP), increased calcium entry and increased contractility. Electrical properties of the mouse heart are very different from those of dog heart, but if a similar system existed in mouse, it could be uniquely studied through genetic manipulations. To investigate the presence of a RAS in mouse, we measured APs and Ito in isolated myocytes. Application of angiotensin II (A2) for 2 or more hours reduced Ito magnitude, without affecting voltage dependence, and prolonged APs in a dose-dependent manner. Based on dose-inhibition curves, the fast and slow components of Ito (Ito,fast and IK,slow) appeared to be coherently regulated by [A2], with 50% inhibition at an A2 concentration of about 400 nM. This very high K0.5 is inconsistent with systemic A2 effects, but is consistent with an autocrine RAS in mouse heart. Pre-application of the microtubule destabilizing agent colchicine eliminated A2 effects on Ito and AP duration, suggesting these effects depend on intracellular trafficking. Application of the biased agonist SII ([Sar1-Ile4-Ile8]A2), which stimulates receptor internalization without G protein activation, caused Ito reduction and AP prolongation similar to A2-induced changes. These data demonstrate AT1R mediated regulation of Ito in mouse heart. Moreover, all measured properties parallel those measured in dog heart, suggesting an autocrine RAS may be a fundamental feedback system that is present across species.

No MeSH data available.


Related in: MedlinePlus

A2-induced electrical remodeling was blocked in cells pretreated with colchicine for 2 hours prior to A2 treatment.(A) Example traces of action potentials recorded from untreated, 5 μM colchicine treated, 5 μM A2 treated, and both colchicine+A2 treated cells. Repolarization delays were observed in A2 treated myocytes as indicated by prolonged APD50(B) and APD90(C) but these effects were blocked in cells pretreated with colchicine. These results suggest that A2 induced electrical remodeling requires functioning intracellular transport mechanisms and may involve the internalization of AT1Rs, ion channels or both. Based on the students t-test, A2 significantly increased the APD50 (P < 0.05) but the increase in APD90 did not quite reach the standard for significance (P = 0.06). However, given these results and the data in Fig 1, it seems reasonable to suggest that A2 increased AP duration in control, but there is no A2 effect when the cells were pretreated with colchicine.
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pone.0138711.g005: A2-induced electrical remodeling was blocked in cells pretreated with colchicine for 2 hours prior to A2 treatment.(A) Example traces of action potentials recorded from untreated, 5 μM colchicine treated, 5 μM A2 treated, and both colchicine+A2 treated cells. Repolarization delays were observed in A2 treated myocytes as indicated by prolonged APD50(B) and APD90(C) but these effects were blocked in cells pretreated with colchicine. These results suggest that A2 induced electrical remodeling requires functioning intracellular transport mechanisms and may involve the internalization of AT1Rs, ion channels or both. Based on the students t-test, A2 significantly increased the APD50 (P < 0.05) but the increase in APD90 did not quite reach the standard for significance (P = 0.06). However, given these results and the data in Fig 1, it seems reasonable to suggest that A2 increased AP duration in control, but there is no A2 effect when the cells were pretreated with colchicine.

Mentions: Colchicine disrupts vesicular transport by binding to tubulin and inhibiting microtubule polymerization. Fig 5 presents the effect of colchicine on A2-induced electrical remodeling in mouse myocytes. Fig 5A shows representative APs from untreated (Control), colchicine treated, A2 treated, and both colchicine and A2 treated cells. Fig 5B & 5C show APD50 and APD90 measured from the same four groups of cells. APDs in CON cells were significantly shorter than those in A2 treated cells (CON APD50: 4.59 ± 0.64 ms and APD90: 24.33 ± 2.15 ms, n = 6), and (A2 treated cells APD50: 8.61 ± 1.37 ms and APD90: 40.40 ± 7.95 ms, n = 6). Whereas no significant difference was observed between CON and Col treated cells (Col APD50: 4.85 ± 1.05 ms and APD90: 24.65 ± 4.39 ms, n = 6) or between CON and Col+A2 treated cells (Col+A2 treated APD50: 4.29 ± 0.40 ms and APD90: 23.43 ± 5.04, n = 6). Though colchicine alone appears to have no effect on the AP, pre-incubation with colchicine blocks A2 induced changes in AP morphology. Thus inhibition of microtubule assembly and disruption of vesicular trafficking alters the normal activity of AT1Rs in mouse and dog heart [30], possibly by blocking receptor internalization. Another possibility is that Ito,fast and IK,slow internalize upon A2 stimulation under normal conditions and disruption of the microtubule network prevents their internalization. Either possibility is consistent with our data, or perhaps both if receptor and channels are co-internalized upon receptor activation.


Angiotensin II Type 1 Receptor-Mediated Electrical Remodeling in Mouse Cardiac Myocytes.

Kim J, Gao J, Cohen IS, Mathias RT - PLoS ONE (2015)

A2-induced electrical remodeling was blocked in cells pretreated with colchicine for 2 hours prior to A2 treatment.(A) Example traces of action potentials recorded from untreated, 5 μM colchicine treated, 5 μM A2 treated, and both colchicine+A2 treated cells. Repolarization delays were observed in A2 treated myocytes as indicated by prolonged APD50(B) and APD90(C) but these effects were blocked in cells pretreated with colchicine. These results suggest that A2 induced electrical remodeling requires functioning intracellular transport mechanisms and may involve the internalization of AT1Rs, ion channels or both. Based on the students t-test, A2 significantly increased the APD50 (P < 0.05) but the increase in APD90 did not quite reach the standard for significance (P = 0.06). However, given these results and the data in Fig 1, it seems reasonable to suggest that A2 increased AP duration in control, but there is no A2 effect when the cells were pretreated with colchicine.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4591968&req=5

pone.0138711.g005: A2-induced electrical remodeling was blocked in cells pretreated with colchicine for 2 hours prior to A2 treatment.(A) Example traces of action potentials recorded from untreated, 5 μM colchicine treated, 5 μM A2 treated, and both colchicine+A2 treated cells. Repolarization delays were observed in A2 treated myocytes as indicated by prolonged APD50(B) and APD90(C) but these effects were blocked in cells pretreated with colchicine. These results suggest that A2 induced electrical remodeling requires functioning intracellular transport mechanisms and may involve the internalization of AT1Rs, ion channels or both. Based on the students t-test, A2 significantly increased the APD50 (P < 0.05) but the increase in APD90 did not quite reach the standard for significance (P = 0.06). However, given these results and the data in Fig 1, it seems reasonable to suggest that A2 increased AP duration in control, but there is no A2 effect when the cells were pretreated with colchicine.
Mentions: Colchicine disrupts vesicular transport by binding to tubulin and inhibiting microtubule polymerization. Fig 5 presents the effect of colchicine on A2-induced electrical remodeling in mouse myocytes. Fig 5A shows representative APs from untreated (Control), colchicine treated, A2 treated, and both colchicine and A2 treated cells. Fig 5B & 5C show APD50 and APD90 measured from the same four groups of cells. APDs in CON cells were significantly shorter than those in A2 treated cells (CON APD50: 4.59 ± 0.64 ms and APD90: 24.33 ± 2.15 ms, n = 6), and (A2 treated cells APD50: 8.61 ± 1.37 ms and APD90: 40.40 ± 7.95 ms, n = 6). Whereas no significant difference was observed between CON and Col treated cells (Col APD50: 4.85 ± 1.05 ms and APD90: 24.65 ± 4.39 ms, n = 6) or between CON and Col+A2 treated cells (Col+A2 treated APD50: 4.29 ± 0.40 ms and APD90: 23.43 ± 5.04, n = 6). Though colchicine alone appears to have no effect on the AP, pre-incubation with colchicine blocks A2 induced changes in AP morphology. Thus inhibition of microtubule assembly and disruption of vesicular trafficking alters the normal activity of AT1Rs in mouse and dog heart [30], possibly by blocking receptor internalization. Another possibility is that Ito,fast and IK,slow internalize upon A2 stimulation under normal conditions and disruption of the microtubule network prevents their internalization. Either possibility is consistent with our data, or perhaps both if receptor and channels are co-internalized upon receptor activation.

Bottom Line: Pre-application of the microtubule destabilizing agent colchicine eliminated A2 effects on Ito and AP duration, suggesting these effects depend on intracellular trafficking.Application of the biased agonist SII ([Sar1-Ile4-Ile8]A2), which stimulates receptor internalization without G protein activation, caused Ito reduction and AP prolongation similar to A2-induced changes.Moreover, all measured properties parallel those measured in dog heart, suggesting an autocrine RAS may be a fundamental feedback system that is present across species.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology & Biophysics, State University of New York at Stony Brook, Stony Brook, New York, United States of America.

ABSTRACT
We recently characterized an autocrine renin angiotensin system (RAS) in canine heart. Activation of Angiotensin II Type 1 Receptors (AT1Rs) induced electrical remodeling, including inhibition of the transient outward potassium current Ito, prolongation of the action potential (AP), increased calcium entry and increased contractility. Electrical properties of the mouse heart are very different from those of dog heart, but if a similar system existed in mouse, it could be uniquely studied through genetic manipulations. To investigate the presence of a RAS in mouse, we measured APs and Ito in isolated myocytes. Application of angiotensin II (A2) for 2 or more hours reduced Ito magnitude, without affecting voltage dependence, and prolonged APs in a dose-dependent manner. Based on dose-inhibition curves, the fast and slow components of Ito (Ito,fast and IK,slow) appeared to be coherently regulated by [A2], with 50% inhibition at an A2 concentration of about 400 nM. This very high K0.5 is inconsistent with systemic A2 effects, but is consistent with an autocrine RAS in mouse heart. Pre-application of the microtubule destabilizing agent colchicine eliminated A2 effects on Ito and AP duration, suggesting these effects depend on intracellular trafficking. Application of the biased agonist SII ([Sar1-Ile4-Ile8]A2), which stimulates receptor internalization without G protein activation, caused Ito reduction and AP prolongation similar to A2-induced changes. These data demonstrate AT1R mediated regulation of Ito in mouse heart. Moreover, all measured properties parallel those measured in dog heart, suggesting an autocrine RAS may be a fundamental feedback system that is present across species.

No MeSH data available.


Related in: MedlinePlus