Investigation of the cardiovascular effects of apelin
Hamilton-Smith, Katherine Mary
Smith, Katherine Mary Hamilton
Apelin was discovered in 1998 as the endogenous peptide ligand of the orphan APJ receptor. The apelin system is well conserved across vertebrate species and is reported to have cardiovascular effects including positive inotropy, vasodilation, vasoconstriction and cardioprotection during ischaemia. Recent studies in human healthy volunteers and in chronic heart failure patients have highlighted the apelin system as a potential target for drug development. However, the cellular and molecular pathways through which apelin acts remain poorly understood. This study aimed to confirm the inotropic and vasoactive actions of apelin and to further examine the proposed cardioprotective effects of apelin under ischaemic and hypoxic conditions. Cardioprotection is defined as a mechanism, for example induced by a drug, which reduces injury in response to ischaemia or hypoxia. In vivo in the anaesthetised rat, apelin was administered as a bolus dose via the cannulated jugular vein and mean arterial pressure was measured by cannulation of the carotid artery. Pyr-apelin-13 had no effect on heart rate or mean arterial pressure. Apelin-13 decreased mean arterial pressure by approximately 20 mmHg, although the effect was highly variable among animals. Apelin-16 consistently lowered heart rate, but had no effect on mean arterial pressure. In rat isolated mesenteric arteries, studied using wire myography, apelin-13 and apelin-36 had no vasodilator or vasoconstrictor effect. In rat isolated right ventricular papillary muscles and right atrial strips, no change in tension, time to peak or time to relax was observed in response to pyr-apelin-13 despite responding to standard pharmacological stimuli such as noradrenaline and increased calcium concentrations in the bathing medium. In isolated, perfused rat heart (Langendorff), infusion of apelin-16 for 15 minutes did not alter developed pressure, rate of rise or rate of fall detected by an intraventricular balloon positioned in the left ventricle throughout the infusion. As the isolated perfused hearts did not demonstrate an inotropic effect in response to apelin, no cardioprotective studies were carried out in this model. Cardioprotective studies of apelin were performed in zebrafish embryos 3 – 5 days post fertilisation (dpf). I developed a hypoxia-recovery model in which we could test the effect of pharmacological agents, including apelin, on the hypoxia-recovery response. In zebrafish embryos 3 dpf, 2h hypoxia (1% oxygen) reduced heart rate and wall motion amplitude (to approximately 90% of control) and contraction velocity and relaxation velocity (to approximately 80% of control). All parameters recovered during a subsequent 2h in normoxia. Incubation in pyr-apelin-13 for 1h prior to and throughout hypoxia did not affect the depression in heart rate observed on exposure to hypoxia. However, apelin incubation resulted in an improvement in wall motion amplitude and relaxation velocity and a significant improvement in contraction velocity after hypoxia and throughout recovery. Pyr-apelin-13 had no inotropic or chronotropic effect on baseline heart function in embryos 3 dpf or in isolated hearts from embryos. However, apelin knockdown using a morpholino targeting the exon 2/intron 2 boundary of apelin pre-mRNA resulted in a high mortality rate and a severe total body and cardiovascular phenotype, suggesting that endogenous apelin is crucial during development in zebrafish embryos. In order to test pharmacological agents more efficiently, I developed a semi-quantitative scoring method to screen a larger number of embryos in a reduced time period. Heart rate and circulation was defined as normal, reduced or absent after 2h and 4h in hypoxia and during recovery in normoxia. The abundance of apelin and HIF-1α mRNA was measured using quantitative RT-PCR. In zebrafish 5 dpf, a marked decrease in apelin mRNA expression was observed after 4h, but not 2h, hypoxia and this was not accompanied by a change in HIF-1α mRNA expression. In zebrafish 5 dpf, exogenous pyr-apelin-13 did not affect the proportion of embryos with normal heart rate and circulation at any timepoint in this model. However, desferrioxamine (iron chelator) and α-ketoglutarate (metabolite involved in aerobic respiration) significantly increased the proportion of embryos with normal heart rate and circulation during the recovery phase. In summary, apelin-13 and apelin-16 were effective in lowering mean arterial pressure and heart rate, respectively, in the anaesthetised rat. However, apelin-13 did not vasodilate or vasoconstrict rat isolated mesenteric arteries. There was no effect of apelin on contractility parameters in rat isolated papillary muscles or in the isolated, perfused rat heart which made it difficult to pursue a cardioprotective effect in this model. In zebrafish, endogenous apelin appeared to be crucial in the development of the embryo, while exogenous apelin had no inotropic effect on cardiac function. In hypoxia-recovery, we demonstrate a cardioprotective effect of apelin in zebrafish 3 dpf, but not zebrafish 5 dpf.