Role of TRPC-channels in cardiac hyperthrophy and platelet aggregation : Analysis of TRPC-deficient mice

Since its first description it was proposed that members of the TRP ion channel family expressed in platelets, endothelial and cardiac cells are involved in various cardiovascular processes. In this thesis I analyzed the role of TRPC channels in platelet aggregation and in the development of cardiac hypertrophy. Pathological cardiac hypertrophy is an adaptive response of the heart to mechanical load abnormalities, but can also be induced by direct stimulation of G protein coupled receptors. It is characterized by elevated intracellular diastolic Ca levels which initiate Cadependent signaling cascades. The mechanisms causing impaired intracellular Ca homeostasis are still incompletely understood, but receptor-operated Ca channels of the TRPC family might be activated by neurohumoral hypertrophic stimuli and could play a role here. Ca entry from the extracellular space is also critical for platelet activation and aggregation which plays an important role in primary hemostasis. Here, Ca entry follows activation of plasma membrane receptors including ADP receptors and TRPC proteins might contribute to this mode of platelet activation. In this study TRPC6 proteins were identified in heart and platelets from wild type but not from TRPC6-/mice. In RT-PCR experiments transcripts from TRPC1, TRPC3, TRPC4 and TRPC6 were amplified from cardiac fibroblasts; however, only TRPC1 transcripts were amplified from isolated cardiomyocytes. TRPC1 and TRPC6 transcripts were amplified from platelets. Unexpectedly, neurohumoral cardiac hypertrophy induced by isoproterenol and angiotensin II (ATII) was not reduced in TRPC3/TRPC6 (-/-) mice. In contrast, the hypertrophic response was significantly reduced in TRPC1/TRPC4 (-/-) after isoproterenol and ATII treatment, respectively. I could rule out differences in renal renin secretion, basal plasma angiotensinogen levels or in blood pressure regulation during isoproterenol infusion; in addition, the reduced hypertrophy response in TRPC1/TRPC4 (-/-) mice is not due to deletion of either TRPC1 or TRPC4 proteins alone. An impaired ATII-induced signaling in TRPC1/TRPC4 (-/-) mice was observed and this could play a role possible through regulation of heart rate or Ca signaling in cardiac fibroblast. Although the underlying mechanisms are not completely understood, TRPC1 and TRPC4 proteins were identified in this study as positive regulators of the neurohumoral-induced cardiac hypertrophy development. In mice lacking either TRPC1, TRPC5 or TRPC6 and mice lacking both TRPC4 and TRPC6 platelet aggregation was found to be unaltered. In contrast, ADP-, Thromboxane A2-, and thrombininduced aggregation was significantly impaired in TRPC3/TRPC6 and TRPC1/TRPC6 deficient mice, and the reduction in the aggregation response was amplified by inactivating P2Y12 receptors with clopidogrel treatment. In contrast, collagen-induced aggregation was unaffected. Therefore, I propose TRPC1, TRPC3 and TRPC6 proteins as crucial regulators of ADP-induced platelet aggregation through P2Y1 receptor signaling which could complement the collagentriggered pathway that depends on Orai/STIM proteins.