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Redox signalling plays an important role in many aspects of physiology, including that of the cardiovascular system. Perturbed redox regulation has been associated with numerous pathological conditions; nevertheless, the causal relationships between redox changes and pathology often remain unclear. Redox signalling involves the production of specific redox species at specific times in specific locations. However, until recently, the study of these processes has been impeded by a lack of appropriate tools and methodologies that afford the necessary redox species specificity and spatiotemporal resolution. Recently developed genetically encoded fluorescent redox probes now allow dynamic real-time measurements, of defined redox species, with subcellular compartment resolution, in intact living cells. Here we discuss the available genetically encoded redox probes in terms of their sensitivity and specificity and highlight where uncertainties or controversies currently exist. Furthermore, we outline major goals for future probe development and describe how progress in imaging methodologies will improve our ability to employ genetically encoded redox probes in a wide range of situations. This article is part of a special issue entitled “Redox Signalling in the Cardiovascular System.”
Redox signalling is increasingly recognised to play a vital role in physiology. Conversely, its dysregulation has been linked to numerous pathological processes, although cause-effect relationships often remain unclear. Cardiovascular physiology and pathology is no exception to this. On the one hand, it has become clear that physiological processes like cardiomyocyte differentiation and excitation–contraction coupling are under redox control. On the other, aberrant redox signalling seems to be associated with a variety of cardiac pathologies, including arrhythmia and myocardial ischemia-reperfusion. Redox signalling is typically based on the specific and reversible oxido-reductive modification of particular cysteine residues in particular proteins. Redox-regulated proteins have been observed to partake in a wide range of cellular processes including signal transduction, gene expression and metabolism. One specific example with relevance to cardiology is the ryanodine receptor 2 in which the redox state of cysteine residues determines Ca2 + conductivity and thus contributes to the regulation of the cardiac rhythm. The key characteristic of redox signalling is that oxidative and reductive modifications are restricted both spatially and temporally. Under most circumstances, the generation, dispersal and elimination of superoxide and hydrogen peroxide is tightly controlled.
Oxidative reactions are usually confined to specific subcellular compartments or microdomains, and the majority of oxidative and reductive processes are under kinetic control. Catalysts like NADPH oxidases (Nox), localised to specific cellular locations and activated at specific times, allow redox reactions to proceed in the direction of thermodynamic equilibrium only in a confined and transient manner. Elsewhere, kinetic barriers prevent uncontrolled equilibration between the many cellular redox species, at least on biologically relevant time scales. Cells frequently run both oxidative and reductive processes in parallel, even within the same compartment, relying on kinetic and steric separation to prevent thermodynamic equilibration. It therefore makes little sense to ascribe an overall redox state or redox potential to a cell.
A redox potential can only be assigned to a chemically defined redox couple. Importantly, measuring one of these redox couples does not necessarily offer any information regarding the redox state of the other cellular redox couples. Thus, if one seeks to make a global statement about the “cellular redox status” or the “degree of oxidative stress,” the measurement of different redox couples may well lead to very different conclusions. Moreover, the redox state of individual redox couples can differ widely between subcellular compartments, and whole cell measurements do not allow conclusions about individual compartments, e.g. the cytosol.
Taking all of the above considerations into account, it is clear that to better understand redox signalling and its dysregulation, we need to increase the resolution of our measurements. Resolution in this context means both spatiotemporal resolution and chemical resolution. It is imperative that we improve our ability to monitor specific, clearly defined redox species. We must improve our ability to monitor dynamic changes in these redox species with subcellular resolution. This will enable us to move away from poorly defined concepts that are frequently observed in the literature, such as cellular “reactive oxygen species (ROS)” changes or “oxidative stress.”
The last 10 years have seen remarkable progress in our ability to address the issues of redox species specificity and spatiotemporal resolution. A particularly important contribution has been the development of genetically encoded redox probes, which now increasingly allow redox species-specific, dynamic real-time observations, with subcellular resolution in intact living cells, tissues and even whole organisms. Arguably, these developments were initiated by a seminal publication of Jakob Winther and colleagues, describing the development of a redox-sensitive yellow fluorescent protein (rxYFP). This work triggered and inspired further improvements and developments that continue to expand our repertoire of genetically encoded redox probes to this day. We have now reached a stage where rapid redox changes can be observed in living mice, in real-time and with single-organelle resolution. Although progress is obvious, a number of limitations, difficulties and uncertainties remain, and there is still much work to be done.
There have been several detailed reviews on the topic of genetically encoded redox probes, covering most of the relevant aspects, from chemical principles to practical applications. It is not our intention to repeat what has been said already. Here we will discuss some questions relating to the specificity and sensitivity of current and future generations of genetically encoded redox probes. We highlight some of the problems that remain to be solved and offer suggestions as to how this may be achieved. Finally, we give a brief outlook on advancements that we may expect to see in the near future.
Daria Ezeriņa, Bruce Morgan, Tobias P. Dick
