Die Bedeutung der Kalzium/Calmodulin-abhängigen Proteinkinase II für den gestörten Kalziumstoffwechsel der isolierten Rattenherzmuskelzelle unter Doxorubicinbehandlung

Die Bedeutung der Kalzium/Calmodulin-abhängigen Proteinkinase II für den gestörten Kalziumstoffwechsel der isolierten Rattenherzmuskelzelle unter Doxorubicinbehandlung

Ca2+/Calmodulin-dependent protein kinase II contributes to impaired Ca2+ handling properties in isolated rat cardiomyocytes under doxorubicin treatment

Anne Christine Köhler

Doctoral thesis

Date of Examination: 2013-07-08

Date of issue: 2013-07-08

Advisor: Maier, Lars S. Prof. Dr.

Referee: Schild, Detlev Prof. Dr.

Referee: Katschinski, Dörthe Prof. Dr.

Persistent Address: http://hdl.handle.net/11858/00-1735-0000-0001-BAA9-2

Files in this item

Name: Dissertation_Anne_C_Koehler1.pdf

Size: 26,2 MB

Format: PDF

View/Open

The following license files are associated with this item:

Creative Commons

Abstract

English

Doxorubicin (DOX), a member of the anthracycline family, is one of the oldest and most commonly used chemotherapeutic agents. Despite its therapeutic benefits, its cardiotoxic potential, resulting in severe cardiac damage, such as toxic-dilated cardiomyopathy, limits its clinical use. In order to prevent these detrimental side effects, a better understanding of the pathophysiology of DOX-induced cardiotoxicity is essential. The pathomechanism is still poorly understood, although there is some evidence of an involvement of reactive oxygen species (ROS). Ca2+/Calmodulin-dependent protein kinase II (CaMKII), a central regulation enzyme in cardiac Ca2+ handling, mediating i.e. phosphorylation of the cardiac ryanodine receptor (RyR), is known to be activated by an increased level of oxidative stress. Therefore, CaMKII was suspected be a mediator of impaired Ca2+ handling under DOX treatment due to a higher level of [ROS]i. The study was performed on isolated rat cardiomyocytes, using confocal and epifluorescence microscopy to determine [ROS]i and to analyze Ca2+ handling properties. Western blot analysis was used to determine phosphorylation level of CaMKII. Firstly, it could be shown that [ROS]I was increased under DOX treatment. Secondly, diastolic [Ca2+]I was elevated, whereas Ca2+ transient amplitude was diminished. Ca2+ content of the sarcoplasmic reticulum (SR) was also reduced. In accordance, diastolic Ca2+ spark frequency as a possible cause of SR Ca2+ depletion, was elevated under DOX treatment. In line with these findings, CaMKII phosphorylation was increased. Subsequently, pharmacological inhibition of CaMKII was performed, using the CaMKII inhibitors KN-93 and autocamtide-2-related inhibitory peptide (AIP). DOX induced diastolic Ca2+ overload could be reduced, as well as diastolic Ca2+ spark frequency. Furthermore, Ca2+ transient amplitude and SR Ca2+ content could be partially restored. In summary, Ca2+handling properties seem to be critically affected under DOX treatment, presumably due to a prolonged and excessive activation of CaMKII as a result of increased [ROS]i. CaMKII inhibition was sufficient to partly restore Ca2+ homeostasis. Further studies may be needed to determine the definitive role of CaMKII activation in DOX induced cardiotoxicity and to possibly identify new therapeutic strategies.

Keywords: CaMKII; Doxorubicin; EC-coupling

Schlagwörter: Kalzium/Calmodulin-abhängige Proteinkinase II; Doxorubicin; Elektromechanische Kopplung