PDE4-mediated cAMP signalling

cAMP is the archetypal and ubiquitous second messenger utilised for the fine control of many cardiovascular cell signalling systems. The ability of cAMP to elicit cell surface receptor-specific responses relies on its compartmentalisation by cAMP hydrolysing enzymes known as phosphodiesterases. One family of these enzymes, PDE4, is particularly important in the cardiovascular system, where it has been extensively studied and shown to orchestrate complex, localised signalling that underpins many crucial functions of the heart. In the cardiac myocyte, cAMP activates PKA, which phosphorylates a small subset of mostly sarcoplasmic substrate proteins that drive β-adrenergic enhancement of cardiac function. The phosphorylation of these substrates, many of which are involved in cardiac excitation-contraction coupling, has been shown to be tightly regulated by highly localised pools of individual PDE4 isoforms. The spatial and temporal regulation of cardiac signalling is made possible by the formation of macromolecular “signalosomes”, which often include a cAMP effector, such as PKA, its substrate, PDE4 and an anchoring protein such as an AKAP. Studies described in the present review highlight the importance of this relationship for individual cardiac PKA substrates and we provide an overview of how this signalling paradigm is coordinated to promote efficient adrenergic enhancement of cardiac function. The role of PDE4 also extends to the vascular endothelium, where it regulates vascular permeability and barrier function. In this distinct location, PDE4 interacts with adherens junctions to regulate their stability. These highly specific, non-redundant roles for PDE4 isoforms have far reaching therapeutic potential. PDE inhibitors in the clinic have been plagued with problems due to the active site-directed nature of the compounds which concomitantly attenuate PDE activity in all highly localised “signalosomes”

Requirement for sphingosine kinase 1 in mediating phase 1 of the hypotensive response to anandamide in the anaesthetised mouse

In the isolated rat carotid artery, the endocannabinoid anandamide induces endothelium-dependent relaxation via activation of the enzyme sphingosine kinase (SK). This generates sphingosine-1-phosphate (S1P) which can be released from the cell and activates S1P receptors on the endothelium. In anaesthetised mice, anandamide has a well-characterised triphasic effect on blood pressure but the contribution of SK and S1P receptors in mediating changes in blood pressure has never been studied. Therefore, we assessed this in the current study. The peak hypotensive response to 1 and 10 mg/kg anandamide was measured in control C57BL/6 mice and in mice pretreated with selective inhibitors of SK1 (BML-258, also known as SK1-I) or SK2 ((R)-FTY720 methylether (ROMe), a dual SK1/2 inhibitor (SKi) or an S1P1 receptor antagonist (W146). Vasodilator responses to S1P were also studied in isolated mouse aortic rings. The hypotensive response to anandamide was significantly attenuated by BML-258 but not by ROMe. Antagonising S1P1 receptors with W146 completely blocked the fall in systolic but not diastolic blood pressure in response to anandamide. S1P induced vasodilation in denuded aortic rings was blocked by W146 but caused no vasodilation in endothelium-intact rings. This study provides evidence that the SK1/S1P regulatory-axis is necessary for the rapid hypotension induced by anandamide. Generation of S1P in response to anandamide likely activates S1P1 to reduce total peripheral resistance and lower mean arterial pressure. These findings have important implications in our understanding of the hypotensive and cardiovascular actions of cannabinoids

Small-molecule allosteric activators of PDE4 long form cyclic AMP phosphodiesterases

Cyclic AMP (cAMP) phosphodiesterase-4 (PDE4) enzymes degrade cAMP and underpin the compartmentalization of cAMP signaling through their targeting to particular protein complexes and intracellular locales. We describe the discovery and characterization of a small-molecule compound that allosterically activates PDE4 long isoforms. This PDE4-specific activator displays reversible, noncompetitive kinetics of activation (increased Vmax with unchanged Km), phenocopies the ability of protein kinase A (PKA) to activate PDE4 long isoforms endogenously, and requires a dimeric enzyme assembly, as adopted by long, but not by short (monomeric), PDE4 isoforms. Abnormally elevated levels of cAMP provide a critical driver of the underpinning molecular pathology of autosomal dominant polycystic kidney disease (ADPKD) by promoting cyst formation that, ultimately, culminates in renal failure. Using both animal and human cell models of ADPKD, including ADPKD patient-derived primary cell cultures, we demonstrate that treatment with the prototypical PDE4 activator compound lowers intracellular cAMP levels, restrains cAMP-mediated signaling events, and profoundly inhibits cyst formation. PDE4 activator compounds thus have potential as therapeutics for treating disease driven by elevated cAMP signaling as well as providing a tool for evaluating the action of long PDE4 isoforms in regulating cAMP-mediated cellular processes

Characterising the phosphorylation and SUMOylation of cardiac troponin I in heart failure

Troponin I (TnI) is the inhibitory subunit of the troponin (Tn) complex of the myofilament. TnI’s function is regulated by phosphorylation at a number of sites by different kinases. As a result of β-adrenergic stimulation, protein kinase A (PKA) phosphorylates TnI at Ser23/24, which causes positive lusitropy and inotropy. The PKA-mediated phosphorylation events on cardiac excitation contraction coupling proteins are often controlled by phosphodiesterase (PDE) enzymes which degrade cyclic 3’,5’-adenosine monophosphate (cAMP), reducing local activation of PKA. This is accomplished by the integration of PDEs into macromolecular complexes, or signalosomes, containing PKA, PKA substrate proteins, anchoring proteins and often other kinases and phosphatases. Research published in another thesis from the Baillie lab indicated that PDE4D9 binds directly to TnI, thereby regulating the cAMP dynamics at the myofilament and the PKA phosphorylation events. Little is known about other post-translational modifications of TnI, but the identification of other modifications could lead to better understanding of the regulation of TnI function within the myofilament and how this is altered in disease states. SUMOylation is a post-translational modification in which a small ubiquitin-like modifier (SUMO) is covalently attached to a substrate protein by an enzymatic cascade similar to the ubiquitination cascade. This thesis began with the testing of a disruptor peptide which was designed to interrupt the proposed interaction between PDE4D9 and TnI. It was hypothesised that disruptor peptides would ‘unhook’ the PDE4D9-TnI complex, allowing enhanced cAMP dynamics at the myofilament and enhanced PKA phosphorylation of TnI. However, using fluorescence resonance energy transfer (FRET) and immunoblotting for phosphorylation levels, it was shown that disruption of the proposed interaction did not significantly affect the outcomes of β-adrenergic signalling at the myofilament. Subsequent attempts to confirm the existence of the proposed interaction were unsuccessful suggesting that PDE4D9 may not be a TnI binding partner after all. Further study is necessary to determine the mechanisms by which PDEs regulate signalling at the myofilament. In silico analysis of TnI revealed a high probability SUMOylation site (K177) suggesting that TnI could be a SUMO substrate. This hypothesis was tested in the second part of this thesis. For the first time, it was shown that TnI can be SUMOylated using a number of biochemical techniques. Furthermore, detection of SUMOylated TnI was facilitated by the successful development of a SUMO-TnI site-specific antibody, the first of its kind to detect a SUMOylated substrate protein. Interestingly, the levels of SUMOylated TnI were significantly enhanced in human heart disease, suggesting a role in the pathophysiology of disease. Functional analyses of the role of TnI SUMOylation in protein stability and myofilament dynamics were then carried out. Viral overexpression of mutant TnI in which the SUMO acceptor lysine was mutated did not have any effect on TnI stability nor the contractility of neonatal rat ventricular myocytes (NRVM). However, functional differences were discovered when mutant TnI was overexpressed in adult rabbit ventricular myocytes (ARVM) and SUMOylation was globally upregulated. The data suggested that TnI SUMOylation may have a scavenging effect, sequestering SUMO proteins from other myofilament SUMO targets. The present work provides a major contribution to the field, showing for the first time that TnI can be modified by SUMOylation and that the modified protein alters myofilament function. Understanding the way post-translational modifications affect the function of cardiac excitation-contraction coupling proteins is necessary for a full understanding of pathophysiology, especially when the modification has been shown to be altered in disease states. Further work elucidating the molecular mechanisms by which TnI SUMOylation alters myofilament dynamics may reveal potential therapeutic targets for heart disease

SUMOylation does not affect cardiac troponin I stability but alters indirectly the development of force in response to Ca2+

Post-translational modification of the myofilament protein troponin I by phosphorylation is known to trigger functional changes that support enhanced contraction and relaxation of the heart. We report for the first time that human troponin I can also be modified by SUMOylation at lysine 177. Functionally, TnI SUMOylation is not a factor in the development of passive and maximal force generation in response to calcium, however this modification seems to act indirectly by preventing SUMOylation of other myofilament proteins to alter calcium sensitivity and cooperativity of myofilaments. Utilising a novel, custom SUMO site-specific antibody that recognises only the SUMOylated form of troponin I, we verify that this modification occurs in human heart and that it is upregulated during disease