Role of Secretory Processes in Cardiac Fibroblasts for Heart Failure Development and Progression
by Naim Kittana
Date of Examination:2014-11-18
Date of issue:2014-11-27
Advisor:Prof. Dr. Susanne Lutz
Referee:Prof. Dr. Susanne Lutz
Referee:Prof. Dr. Blanche Schwappach
Referee:Prof. Dr. Jürgen Brockmöller
Persistent Address:
http://dx.doi.org/10.53846/goediss-4799
Files in this item
Name:Naim Kittana, PhD Thesis.pdf
Size:3.74Mb
Format:PDF
Description:Doctoral Thesis
The following license files are associated with this item:
Abstract
English
Cardiac fibroblasts play a major role in fibrogenesis associated with heart failure, since they produce ECM components and secrete important fibrosis-associated mediators, such as connective tissue growth factor (CTGF). CTGF expression and secretion can be induced by the angiotensin II (Ang II) type1 receptor (AT1-R) activation. Within this thesis, the role of the Ca2+- and cytoskeleton-dependent signaling pathways elicited by Ang II on the regulation of CTGF were investigated in neonatal rat cardiac fibroblasts (NRCF). Ang II was shown to induce a Ca2+ transient via the PLC-β canonical pathway with an EC50 in a physiological range. This transient was detectable in the absence of extracellular Ca2+ and in accordance, the depletion of intracellular Ca2+ by thapsigargin (TGN) completely suppressed it. Interestingly, the blockade of the transmembrane TRPC3 channels by pyrazole 3 increased this transient and strongly inhibited the Ang II-induced Ca2+ oscillations. In addition, the Ca2+ oscillation could be also blocked by TGN. Moreover, the NADPH oxidase and its accessory activator Rac1 were found to be involved in the regulation of the induced Ca2+ transient in the cytosol and in mitochondria. In the next step, the impact of Ca2+ and its effector proteins were studied on CTGF expression and secretion. First, to demonstrate the overall outcome of Ca2+ on the regulation of CTGF, BAPTA-AM was used and showed that the chelation of intracellular Ca2+ resulted in the inhibition of CTGF expression and secretion. Second, with help of the inhibitor Go 6983 the involvement of protein kinase C (PKC) as a downstream mediator could be established. Third, in contrast to the downregulation of CTGF expression by PKC inhibition, an increase in CTGF expression was found in response to calcineurin inhibition by cyclosporin A (CsA). Next, the role of the actin cytoskeleton and of microtubules were studied. Actin filament disruption by latrunculin A (LAT-A) led to a similar decrease in CTGF expression as BAPTA-AM, which also had an actin depolymerizing effect. Similar, PKC inhibition was found to disrupt the actin cytoskeleton. All three interventions led in addition to major morphological changes of the Golgi apparatus, which is the major storage place of CTGF. In contrast to the actin cytoskeleton, the integrity of the microtubules was not affected under most conditions. To finally determine the role of these thick filaments in CTGF regulation, the microtubules depolymerizing drug colchicine was applied, which dispersed the Golgi apparatus, increased the CTGF expression and uncoupled it from the AT1R signaling cascade. In summary, CTGF is regulated by a crosstalk of Ca2+-dependent signaling, involving PKC and calcineurin, and cytoskeleton-dependent pathways involving actin filaments and microtubules.
Keywords: Fibrosis; Angiotensin II; Heart failure; Cardiac fibroblasts; Connective tissue growth factor (CTGF); Secretion; Calcium signaling; cytoskeleton-dependent signaling; Protein kinase C (PKC); Calcineurin; Actin; Microtubules; Live cell calcium imaging; Calcium oscillation; Calcium transient; TRPC3 channels
