Abstract
Background and aims: Increased circulating soluble urokinase plasminogen activator receptor (suPAR) levels are associated with adverse cardiovascular outcomes, however, any cardiovascular-related functions remain unknown. This study aimed to (i) document haemodynamic profiles in isolated rat hearts under the influence of suPAR, and to (ii) explore any mechanisms triggered by suPAR following myocardial ischaemia-reperfusion.
Methods: We undertook a 4-pronged approach (Figure 1). A Langendorff isolated rat heart model was used to perfuse all hearts with Krebs-Henseleit solution flowing retrogradely through the aorta at 37°C. A balloon inserted into the left ventricle allowed measurements of ventricular pressure, and a pressure transducer placed above the aorta recorded perfusion pressure for coronary flow rates. The experimental protocol consisted an initial 30-minute stabilisation period, followed by 40 minutes of ischemia via buffer flow cessation. Hearts were subsequently recovered during 90 minutes of reperfusion, receiving either suPAR treatment or perfusion buffer as controls. An inhibitor arm comprising co-infusion of suPAR and a monoclonal antibody capable of binding to suPAR was also assessed. Troponin T was measured in outflow perfusates collected at specific intervals. Proteins from hearts in treatment groups were separated using gel electrophoresis, with Western blotting to visualise the phosphorylation status of kinases (Akt, STAT). High-flow respirometry and fluorometry were investigated in cardiac mitochondria to determine respiration rates and ATP production in ischaemia reperfusion under the influence of suPAR. Statistical analysis was conducted with ANOVA using SPSS.
Results: Hearts receiving suPAR infusion at reperfusion (n = 16) showed a significant increase in developed pressure (p = .017) and lower perfusion pressure (p = .02) compared to controls (n = 16), revealing that suPAR-treated hearts recovered with better contractility and vasodilatory properties post-ischaemia. suPAR’s haemodynamic profiles in the antibody group were similar to controls, attenuating suPAR-induced effects (n = 8). Outflow effluents in suPAR-treated hearts obtained >3 fold lower serial Troponin T values than controls (p < .001). Heart tissues receiving suPAR treatment revealed a 1.7-fold increase in Akt (p = .001) and a 3.8-fold increase in STAT3 phosphorylation compared to controls (p < .001) and the inhibitor group (p = .04). Finally, suPAR increased mitochondrial ATP production by 28% (p = .013) compared to controls after ischaemia.
Conclusion: This is a world-first demonstration of suPAR’s cardioprotective influences in isolated rat hearts recovering from ischaemia. suPAR’s ability to promote cardiac contractility and vasodilation was associated with lesser injury as shown by Troponin T reduction. Mechanistically, suPAR infusion upregulated two key cardioprotective pathways, which may also function to protect cardiac mitochondria.Figure 1 - Schematic of Methods