INTACT HEARTS: A NEW TOOL FOR ELUCIDATING THE PHYSIOLOGY COF THE PRION PROTEIN

The elusive function of PrPC hampers the understanding of the molecular mechanism at the basis of prion diseases, and the development of suitable therapeutic protocols. Use of cell model systems, and genetically modified animals, have nevertheless suggested a number of potential roles for the protein, ranging from cell survival to differentiation. Because we now know that muscle is involved in PrPC pathophysiology, we have considered intact heart paradigms for the in situ study of the cell-protecting function of PrPC. Isolated muscle organs retain the cell native environment and are also more suitable to experimental designs than whole animals. Accordingly, by taking advantage of mice expressing different PrPC amounts (WT, KO and overexpressors(OE)), the protection of PrPC against cell oxidative injuries was investigated in isolated hearts subjected to ischemia/reperfusion protocols that involve oxidative stress. In line with the putative capability of PrPC to antagonize oxidative injury and cell death mechanisms, our prediction was that hearts from adult PrPC-KO mice manifest an overt phenotype after ischemic challenge, resulting in exacerbation of heart oxidative damage. Conversely, PrPC overexpressing mice should demonstrate a higher resistance over ROS production. Myocardial viability was assessed by lactic dehydrogenase (LDH) release into the coronary effluent, while quantification, by immunoblot assays, of myocardial damage was based on myofibrillar protein oxidation. We found that in PrPC-OE hearts 15 min-reperfusion after 40 min of no-flow ischemia was associated with a lower LDH release, compared to hearts from KO and WT mice. We then reasoned that absence of PrPC should increase the effect of ischemic preconditioning (IPC), in contrast to the less evident protection in hearts from PrPC-OE mice, i.e. PrPC acts as an antioxidant. Indeed, our data on hearts subjected to IPC nicely fits with this prediction, given that IPC led to a strong decrease of damage in PrPC-KO hearts, an intermediate protection in WT hearts, and no significant effect in PrPC-OE hearts. We also applied protocols of non-ischemic oxidative injury, by subjecting isolated hearts to perfusion with hydrogen peroxide. Such treatment was associated with a significantly larger LDH release in PrPC-KO hearts, compared to hearts from WT and OE mice. The protection by PrPC over ROS damage was also evident from the myofibrillar (tropomyosin) oxidation pattern of the hearts isolated from the different animals, under the used experimental protocols. This data thus supports both the value of the in situ muscle paradigm for studying the physiologic function of PrPC, and the role of PrPC against oxidative insults.