Reactive oxygen species (ROS) are known to play a central role in the pathology of acute myocardial infarction (AMI), where their production is driven by the metabolism of the citric acid cyclic intermediate succinate that accumulates during ischaemia. This is a major driver of damage upon reperfusion of ischaemic tissue. ROS are however increasingly understood to not only have negative effects, but also to act as biological signalling molecules that are able to activate protective pathways. In order to investigate how such antagonistic processes might interplay in the setting of ischaemia/reperfusion injury (IRI), it is necessary to have a tool that allows the titration of a precise amount of ROS, ideally occurring in a manner that closely mimics endogenous ROS production in both location and the exact reactive species. To this end we validate that the mitochondria-targeted compound MitoPQ produces ROS in this manner and also develop a bespoke control compound that matches the molecular properties of MitoPQ closely with the exception that it does not redox cycle at complex I in the mitochondrial respiratory chain. These compounds are then used in model systems in vitro and in the mouse in vivo to show that whilst high doses of exogenous ROS are damaging to the heart undergoing ischaemia/reperfusion, low doses protect the heart against this insult as shown by smaller infarct sizes. Such a biphasic relationship is also seen between mitochondrial ROS and a range of cellular functions, and further allow us to demonstrate clearly for the first time that a primary increase in mitochondrial ROS may result in these changes rather than the other way around. We also validate two further tools for use in vivo that enable the investigation of the effects of changes in mitochondrial redox status. MitoNeoD is an improved probe for the detection of mitochondrial superoxide using the exomarker approach, while MitoCDNB allows the selective disruption of mitochondrial thiol redox state. The metabolic changes occurring in IRI are also investigated, applying mass spectrometry and magnetic resonance spectroscopy to measure the accumulation of succinate during ischaemia and show that the existing cardioprotective strategies of ischaemic preconditioning and moderate cooling do not alter the extent to which succinate is accumulated during ischaemia. The use of disodium malonate to inhibit the metabolism of succinate at reperfusion is conversely found to protect the heart against IRI. A pilot experiment is also conducted to validate a protocol of volitional exercise post AMI that has been reported to have beneficial effects on heart function during the development of heart failure with a view to investigating a potential role of succinate, but no difference is observed between exercised and sedentary mice in our hands.