Identification de nouveaux mécanismes régulateurs des pulsars calciques endothéliaux d’artères mésentériques de souris

Endothelial cells (EC) are an important component of the vascular homeostasis. They modulate various vascular functions as vascular tone. The regulation of the smooth muscle cells (SMC) contractile state by the endothelium is multifactorial. Among those various factors, nitric oxide (NO) is identified as a potent vasodilator and the endothelium-derived hyperpolarizing factor (EDHF), which modulates vascular tone by controlling smooth muscle membrane potential. Interestingly, literature shows that both mechanisms involved in endothelial function strongly rely on intracellular calcium (Ca2+) concentration. Moreover, endothelial dysfunction is highly correlated with Ca2+ dyshomeostasis as observed in arterial hypertension (AH). In this line of thought, it’s crucial to extend our knowledge on how intracellular calcium signalling is regulated. During the past 10 years, several studies have revealed the important role of targeting specific cellular function by local Ca2+ signals. Those evidences rely on the significant improvement in imaging technology. Recently, a new localized Ca2+ signalling pathway called Ca2+ pulsars has been described in native endothelial cells from mouse mesenteric resistance arteries. Ca2+ pulsars are a limited, spontaneous and oscillatory Ca2+ release from IP3 receptor (IP3R) located on the endoplasmic reticulum membrane within the myoendothelial projections (MEP). The MEP structure is highly interesting because it constitutes the only site where EC and smooth muscle cell (SMC) from the vascular wall can physically interact. It’s now largely accepted that MEPs act as microdomains for local Ca2+ signals and Ca2+-depend effectors implicated in the SMC contractile state regulation.  Ca2+ pulsars have been shown to be able to modulate Ca2+-activated potassium channel (KCa) located on the plasma membrane of the MEP. Once activated, the K+ efflux will generate the endothelial membrane hyperpolarization, which once transmitted to the underlying SMC will induce their relaxation. Despite the identification of Ca2+ pulsars targeted pathways, little is known about how this local Ca2+ signal is regulated.  A previous study from the laboratory of Dr. Ledoux has shown an alteration in Ca2+ pulsars dynamics in the pathological condition of AH. Consequently, it was essential to extend our knowledge on Ca2+ pulsars regulatory mechanisms to be able to identify the possible pathways implicated into this pathological alteration. In this thesis, two potential regulatory pathways have been investigated. In the first study, the implication of CaMKII, a kinase known to act as a Ca2+ sensor, was assessed by showing various activation states of the kinase depending on Ca2+ oscillation frequencies. We showed that CaMKII might be involved in a negative feedback loop according to its activation pathway. Inhibition of CaMKII in presence of KN-93 showed an increase in the number of Ca2+ pulsars active sites suggesting that CaMKII can control which Ca2+ pulsars site can be activated. This first study demonstrated that CaMKII may regulate the number of Ca2+ pulsars active sites by modulating IP3Rs activity. In the second study, we evaluated if the mitochondria could influence Ca2+ pulsars pattern. The rationale behind the choice of this potential regulatory mechanism is that mitochondria can act as a Ca2+ barrier in different cell types. We showed that mitochondria are preferential localized in front of the MEP in mouse mesenteric arteries. Furthermore, we demonstrated that regulation of Ca2+ pulsar dynamics by mitochondria is multifactorial. This organelle is involved in Ca2+ pulsars dispersion by its ability to reuptake intracellular Ca2+. In addition, mitochondria seems to be important to set the Ca2+ pulsar activity level probably by modulating the Ca2+ environment around the IP3Rs within the MEP. The impact on Ca2+ pulsars activity level may be mediated by mitochondria Ca2+ efflux mechanisms. In both studies presented in this thesis, the two mechanisms (CaMKII and mitochondria) have been studied in physiological conditions without any Ca2+ pulsars stimulation suggesting their role in basal control of Ca2+ pulsars dynamics. Thereby, both studies have filled a gap of knowledge regarding how the Ca2+ pulsar stochasticity is established in physiological conditions. This work brings out new avenues to investigate in endothelial dysfunction associated with intracellular Ca2+ homeostasis alteration.

Keyphrases: Calcium, CaMKII, Vascular endothelium, Myoendothelial projection, Calcium pulsar, Vascular tone, Mitochondria