B-Type Natriuretic Peptide Signal Peptide in Cardiovascular Disease

Abstract

The thesis presented is based on the study of a novel peptide identified in the circulation, B-type natriuretic peptide signal peptide (BNPsp). Signal peptides (SP) are important amino acid sequences in protein synthesis allowing the newly synthesised protein to enter its correct location within the endoplasmic reticulum (ER). The existing protein synthesis paradigm is that once the nascent protein has entered the ER, the SP is destroyed intracellularly. This thesis documents the first evidence that a fragment of BNPsp exists in the peripheral circulation. In targeting the SP of BNP, a well-described cardiac biomarker, the natural assumption would be that this would be found in higher concentrations across the heart and may be released by similar stimuli to the parent protein. A specific radioimmunoassay (RIA) was developed directed towards the C terminal of the 26 amino acid signal peptide. The RIA was validated with the use of high performance liquid chromatography and subsequent mass spectrometric analysis, confirming the identity of BNPsp(17-26) as an authentic entity in the human circulation. Studies detailed in this thesis investigates BNPsp as a biomarker as well as a potential therapeutic for ischaemia reperfusion injury (IRI). As a potential biomarker, we have shown that in the context of ST elevation myocardial infarction (STEMI), peaks of BNPsp are reached early and preceded that of other early markers such as myoglobin. The peaks were short-lived with levels normalising within hours. BNPsp had a release pattern distinct from that of NT-proBNP that peaked at 1-2 days. BNPsp peaked earlier than troponin T as assessed with high sensitivity assays (hsTnT) in dobutamine stress echocardiography (DSE) in both healthy volunteers and those with ischaemic heart disease. NT-proBNP levels in contrast, showed minimal change in the 4 hour sampling period. In this model of reproducible cardiac injury, the levels of BNPsp again normalised rapidly after the DSE, in contrast with hsTnT. In a group of patients with decompensated heart failure, the levels of BNPsp remained within the normal range. In reviewing results from all clinical studies, it is apparent that mechanisms for release and clearance of BNPsp differ from BNP or NT-proBNP. With regards to biological activity, the administration of synthetic human BNPsp(17-26) as a preconditioning agent prior to a forty minute global ischaemic insult in the Langendorff isolated rat heart model exerted a dose-dependent protective effect with improved haemodynamics, marked attenuation of ischaemic damage as determined histologically and 90%+ reduction in the release of troponin/ myoglobin. Protective effects were also seen when the peptide was administered at the time of reperfusion. Negative effects were seen at levels 10-30 times higher than the therapeutic dose. The cardioprotective effects reflected in reduction in infarct size were replicated in an ovine in vivo model of experimental infarction with no adverse haemodynamic effects or signs of toxicity, confirming BNPsp as a potential therapeutic agent in IRI. In summary, these studies have demonstrated that BNPsp has potential to act as an early and dynamic biomarker of cardiac ischaemia as well a potential therapeutic in IRI.