Involvement of Novel Cardiac Peptides in Healthy and Ischemic Hearts
Thesis DisciplineBiological Sciences
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
The role and functions of Urotensin II (UII), Urotensin II-related peptide (URP) and proangiotensin-12 (PA12) are currently ambiguous, either due their relatively new identification and isolation from their host species, or due to contrasting and conflicting reports observing the physiological and pathophysiological role of these spasmogens within the mammalian cardiovascular system. Accordingly, we sought to determine the true physiological functions of these peptides in both healthy and diseased states. The initial task was to reveal potential reasons for the contrasting responses to UII, and to define the role of UII within the isolated rat heart. UII and URP retain a highly conserved cyclic region, shown to be necessary in receptor binding and activation, with the high inter-species variance within the N-terminus reported to be of little importance. Our research revealed UII to be highly species-specific, stimulating potent, sustained vasodilation of the coronary arteries in response to the native form infused, while non-native UII peptides had either no effect, or caused significant vasoconstriction. UII-induced vasodilative effects were found to be mediated by nitric oxide and prostaglandin activity combined. Reviewing publications to date it was evident that many studies employed UII foreign to the host species, reporting potentially untrue effects, based on our findings. Recent studies have identified UII as a potent agent in developing and promoting atherosclerosis and coronary artery disease through UII-induced mitogenic activity and promoting foam cell formation. Hence, we observed the effect of infusing the native species of UII and URP into a model of cardiac ischemia-reperfusion. Both preconditioning the heart with UII or URP, or infusing UII or URP upon reperfusion caused significant coronary vasodilation following ischemia, and significantly attenuated ischemic-induced myocardial injury. These studies indicated elevating UII and URP provided a level of cardioprotection, not only when administered into healthy hearts prior to an ischemic event, but also in hearts having already undergone ischemia and the resultant endothelial damage. PA12 was the third peptide tested in the current thesis. Being newly identified and suggested to be a new component of the renin-angiotensin system (RAS) it was important to define the physiological role of PA12 upon the cardiovasculature, as the RAS is heavily associated with the development and progression of cardiovascular disease. Utilising the Langendorff isolated rat heart technique, PA12 was found to cause potent vasoconstriction of the coronary arteries, mediated by the angiotensin II type 1 receptor (AT₁R). Furthermore, using subjecting the perfusate samples to radioimmunoassay and RP-HPLC revealed PA12 was converted to AngII. Both PA12-induced vasoconstriction and generation of AngII were found to be dependent upon chymase activity, with inhibition of ACE1 having little effect. Myography was employed to further study the vascular response to PA12 throughout the rat arterial system from the common carotid to the femoral arteries. PA12-induced vasoconstriction displayed a potency gradient, with greatest constriction observed in vessels closest to the heart, with potency reduced and eventually lost further from the heart. PA12-induced vasoactivity was shown to be dependent upon both chymase and ACE1 activity, with ACE1 regulating PA12 activity with greater potency. The intracellular pathways stimulated in response to PA12 were defined using western blotting, with PA12 stimulating phosphorylation of ERK1/2, JNK, p38 and PKCα/β₁₁, but having no influence on PKCδ/θ. Stimulation of these pathways is consistent with the observed PA12-induced vasoconstriction, and also indicates that PA12 activation of AT₁R and the subsequent cytokines, could potentially stimulate hypertrophy, apoptosis, cell growth and differentiation, and inflammation, promoting cadiovascular remodelling and progressing atherosclerosis, hypertension and other vascular diseases if not sufficiently regulated. Taken together, these studies indicate PA12 may have a primary role within the local, tissue-based RAS, providing an alternate substrate to angiotensin I, while ACE1 is the primary regulatory enzyme within the circulation. Our findings also display the chymase-dependent PA12/AT₁R pathway as potential novel targets for pharmacological inhibition of RAS activity to ameliorate hypertension and maladaptive vascular remodelling.