AIMSThe aim of this study was to observe the direct physiological and biochemical cardiac effects in response to a newly identified putative component of the renin-angiotensin system, proangiotensin-12 (PA12); and investigate whether PA12 can serve as a substrate for Angiotensin II (AngII) generation.METHODS AND RESULTSThe direct cardiac actions of PA12 and its role as a substrate for chymase-dependent AngII generation were investigated in Sprague-Dawley rats using an isolated heart model of cardiac ischaemia-reperfusion injury. PA12 potently constricted coronary arteries with no significant effect on left-ventricular contractility. PA12 impaired recovery from global ischaemia, maintaining coronary constriction and markedly increasing release of creatine kinase and troponin I (TnI), indicating greater myocardial injury. Analysis of perfusate collected after transcardiac passage revealed a marked increase in AngII production from hearts infused with PA12. Cardiac AngII production was not blocked by angiotensin-converting enzyme inhibitors, whereas inhibition of chymase with chymostatin significantly reduced AngII production and attenuated PA12-induced vasoconstriction and myocardial damage following ischaemia. Furthermore, Angiotensin II type 1 receptor (AT(1)R) blockade abolished PA12 activity. In vitro, PA12 was efficiently and precisely converted to AngII as assessed on reverse phase-high performance liquid chromatography coupled to tandem mass spectrometry. This conversion was blocked by chymostatin.CONCLUSIONPA12 may act as a circulating substrate for cardiac chymase-mediated AngII production, in contrast to ACE-mediated AngII production from AngI.