Angiotensin II reguliert das Natriumkanal- Öffnungsverhalten über zwei Mechanismen: IP3-Rezeptoren aktivieren die CaMKII und ROS die PKA

Angiotensin II reguliert das Natriumkanal- Öffnungsverhalten über zwei Mechanismen: IP3-Rezeptoren aktivieren die CaMKII und ROS die PKA

Angiotensin II regulates sodium channel gating via two mechanisms: IP3-receptors activate CaMKII and ROS activate PKA

Hannah Flebbe

Doctoral thesis

Date of Examination: 2017-09-27

Date of issue: 2017-09-19

Advisor: Maier, Lars S. Prof. Dr.

Referee: Katschinski, Dörthe Prof. Dr.

Referee: Meyer, Thomas Prof. Dr.

Persistent Address: http://hdl.handle.net/11858/00-1735-0000-0023-3F06-C

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Abstract

English

Background: In heart failure (HF) disturbed CaMKII-dependent sodium current (INa) and sodium channel gating leads to arrhythmias and contractile dysfunction. Enhanced Angiotensin II (Ang II) signaling has been implicated in the development of HF, and contributes to contractile dysfunction and arrhythmias. Ang II can activate CaMKII by at least two independent pathways, but the mechanisms are not fully understood yet. One pathway involves Ang II-induced activation of NADPH oxidase 2 (Nox 2) resulting in the generation of reactive oxygen species (ROS). It was shown shown that ROS directly oxidize CaMKII leading to prolonged CaMKII activation, arrhythmias and contractile dysfunction. Alternatively, Ang II stimulates inositol trisphosphate receptors (IP3R), which release Ca2+ from stores into the cytsol. This Ca2+ release (via Calmodulin) could also activate CaMKII. On the other hand, it was recently shown that ROS can also oxidize the regulatory subunit I of the cAMP-dependent protein kinase A (PKA), resulting in redox-dependent activation of PKA. Aim of this study was to discover the effect of Ang II on the INa and the sodium channel gating and to identify the underlying mechanisms of the Ang II dependent signaling. Methods: Peak INa, steady-state inactivation, intermediate inactivation (IIM), recovery from inactivation and late INa was measured in isolated ventricular myocytes from mice lacking the gp91phox subunit of Nox 2 (gp91phox-/-) and wildtype (WT) littermates as control with whole-cell patch clamp technique. Results: Incubation of the cells with Ang II significantly increased INa current density. Inhibition of PKA with H89 significantly blunted this Ang II-effect. The increase was also completely absent in gp91phox-/-. The sodium channel gating was influenced by Ang II as well. In WT, Ang II significantly left-shifted steady-state inactivation consistent with less sodium channels available at a given membrane voltage. Furthermore the IIM was significantly enhanced and the recovery from inactivation significantly slowed. Neither knockout of gp91phox nor PKA inhibition with H89 could reverse this Ang II- induced effects on the channel gating. Late INa was significantly enhanced after incubation with Ang II in a Nox2- and PKA-independent manner. Pharmacological inhibition of both CaMKII with AIP and IP3R with 2-APB could completely reverse the Ang II-induced increase. Conclusion: Ang II has intracellular complex effects through at least two different signal cascades. Via an ROS-dependent activation of the PKA, Ang II influences the regulation of the INa current density. However the gating activity of the sodium channel is regulated independently of ROS and PKA. Besides the late INa is regulated by IP3R and CaMKII independent from ROS and PKA. There may be Ca2+ release by the IP3R and associated Ca2+/Calmodulin-dependent activation of the CaMKII.

Keywords: angiotensin II; sodium channel; heart failure; PKA; CaMKII; ROS