THE IMPORTANT ROLE OF AKT IN THE MODULATION OF HEART INOTROPISM THROUGH L-TYPE CALCIUM CHANNELS FUNCTION
Creators: name: RUSCONI, FRANCESCA
Publisher: name: Universit� degli Studi di Milano
Year: 2010

description: The insulin IGF1/Akt signaling pathway has recently been shown to be critical for the regulation of heart function and physiology. Indeed, compelling evidence shows activation of this pathway as one of the most important determinants for the enhancement of cardiac function and physiological growth in athletes, whereas its impairment is considered critical for the development of heart failure (HF). In this doctoral thesis, our aim was to determine the functional role of known and novel key-factors of this pathway to study whether their modulation might be envisaged as therapeutic tool for curing pathological cardiac hypertrophy (CH) and HF. Physiological CH is an adaptive response of the heart to stimuli, such as developmental growth and training and differs markedly from pathological hypertrophy occurring in patients with HF. In this thesis, we demonstrated the involvement of Akt kinase in regulating heart inotropism by modulating L-Type Ca2+ Channel (LTCC) density and function. In a mouse model with inducible and cardiac specific deletion of PDK1, the upstream activator of Akt, we found that the protein stability of the LTCC pore subunit (Cav?1) can be modulated by the kinase. In particular, phosphorylation of the C-terminal coiled coil of the Cav?2 chaperone subunit enhances LTCC protein stability by prevention of PEST-mediated Cav?1 degradation. Subsequently, to determine whether the modulation of this mechanism may be used for the treatment of HF, we studied the fine-regulation of LTCC density and activity by investigating the functional role of Akt-phosphomimetics Cav?2 constructs. Three Akt-phosphomimetic sequences corresponding to the Cav?2 C-terminal coiled coil were identified and shown to protect Cav?1 from protein degradation, through an increase in the number of functional LTCC. Moreover, to establish whether the Akt-dependent phosphorylation of CaV?2 might be a trigger for the recruitment of other protein interacting partners, yeast two-hybrid screenings of human and mouse heart cDNA expression libraries revealed a fold-back interaction of the Akt-phosphorylated-Cav?2 tail with a region of the Cav?2 globular domain. Co-immunoprecipitation experiments confirmed this interaction, while negative results were obtained when Cav?2-WT was used as bait. This provided the proof of concept for a mechanism of action that relies on Akt-dependent phosphorylation. Site-specific mutagenesis in the identified interacting domain confirmed this mechanism. All togheter, we found that the Akt-dependent protective effect on Cav?1 stability might relay on Cav?2 structural rearrangements, which follow the phosphorylated C-terminal coiled coil fold back on its globular domain. In conclusion, results from this doctoral thesis provide further insights into the role of the insulin IGF1/Akt signaling pathway and its role in the modulation of myocardial physiology and HF. These findings may lead to the development of new therapeutical tools that will be useful for the modulation of impaired cardiac contractility in HF.