Investigating the CaMKKβ signalling pathway and its role in the regulation of blood pressure

Abstract

Calcium/Calmodulin dependent protein kinase kinase beta (CaMKKβ) is a serine/threonine kinase involved in the Calcium/Calmodulin dependent protein kinase (CaMK) cascade. In 2005, CaMKKβ was shown to phosphorylate and activate AMP-activated protein kinase (AMPK), a master regulator of metabolism. Activation of AMPK has previously been reported to inhibit vascular smooth muscle myosin light chain (MLC) phosphorylation. The phosphorylation state of MLC in smooth muscle is known to directly control the contractility of blood vessels and the maintenance of blood pressure. This thesis describes the characterisation of the global CaMKKβ knockout (CaMKK KO) mice, as well as the investigation of CaMKKβ signalling pathways involved in blood pressure regulation using CaMKK KO mice in a model of lipopolysaccharide induced septic shock. Septic shock is a syndrome defined by persistently reduced blood pressure despite fluid resuscitation leading to multiple organ failure resulting from overwhelming infection. Using implantable telemetry probes, aortic blood pressure was recorded in age matched male wild-type (WT) and CaMKK KO mice under both basal conditions and during lipopolysaccharide induced septic shock. In mice with global deletion of CaMKKβ, basal blood pressure was found to be increased compared to their WT littermates. In addition, when treated with intraperitoneally injected bacterial lipopolysaccharide, the lack of CaMKKβ appears to confer protection against hypotension, with significantly higher blood pressure seen in CaMKK KO mice, as well as improved mobility and general physiological state, compared to WT mice. To investigate the mechanism for the increased blood pressure in CaMKK KO mice, aorta lysate MLC kinase activity and MLC phosphorylation were measured and found to be increased in lipopolysaccharide treated CaMKK KO mice compared to WT mice. In contrast to a recent independent study, no indication of altered inflammatory response was found in CaMKK KO mice compared to WT littermates following intraperitoneal lipopolysaccharide injection. Ex vivo lipopolysaccharide treatment induced similar levels of nitric oxide production and phagocytosis in primary peritoneal macrophages isolated from WT and CaMKK KO mice. In summary, CaMKKβ appears to be involved in the regulation of blood pressure. Loss of CaMKKβ in mice protects against septic shock through increased phosphorylation of MLC and the maintenance of blood pressure. Currently, septic shock is associated with a high mortality rate despite the available pharmacological treatments; inhibition of CaMKKβ could provide an alternative strategy to assist with the maintenance of blood pressure during sepsis.