Burke, N; (2017) OPA1 as a target for cardioprotection. Doctoral thesis , UCL (University College London).

Abstract

Background: OPA1 is an inner mitochondrial membrane protein that regulates fusion via its 8 isoforms, which are the product of selective cleaving. Upon cellular stress, OMA1 (a zinc metallopeptidase) cleaves the longer isoforms, disrupting OPA1 function and increasing susceptibility to cell death. 1, 10-phenanthroline (a metallopeptidase inhibitor) has been reported to inhibit OMA1 and preserve OPA1 in in vitro studies. Here, it is investigated if this compound can preserve OPA1 in the murine heart, and confer protection against ischemia reperfusion injury (IRI). Naturally occurring OPA1 mutations lead to a condition known as Dominant Optic Atrophy (DOA), which affects retinal ganglion cells causing blindness. The condition leads to a range of pathologies depending on the mutation, and it was investigated here if primary dermal fibroblasts from DOA patients were more susceptible to simulated ischaemia-reperfusion injury (IRI), and IRI-related insults. Methods and Results: In vivo and ex vivo adult murine hearts, as well as isolated murine cardiomyocytes, were used to investigate OPA1’s protein isoforms, total OMA1 levels, and cell/tissue death in the setting of ischaemia and reperfusion, with and without 1, 10-phenanthroline treatment. Susceptibility to mitochondrial permeability transition pore (MPTP) formation and cristae integrity were also measured. Compared to control, phenanthroline treatment reduced cell/tissue death, delayed MPTP formation, and preserved cristae integrity post IR. DOA patient’s dermal fibroblasts were subjected to H2O2 insults and their susceptibility to form MPTP was measured, as well as assessing mitochondrial morphology. These cells were not significantly more susceptible to H2O2 insults, or MPTP formation, compared to non-DOA control fibroblast, but had alterations in their mitochondrial morphology. Conclusions: It is shown for the first time that OPA1 isoforms are degraded during reperfusion in the heart and that phenanthroline is a cardioprotective agent in the acute setting of cardiac IRI, in part by preserving cristae integrity and delaying MPTP formation. Phenanthroline preserves OPA1 isoforms and reduces total OMA1 at 100 µM. Although phenanthroline is not wholly specific for OMA1, these findings support other studies that highlight the clinical potential of developing a specific small molecule inhibitor of OMA1. The DOA patient fibroblasts investigated are no more sensitive to the IRI-related insults tested compared to control dermal fibroblasts.

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