Oxidative stress plays an important role in the pathogenesis of many disease states. In the heart, reactive oxygen species are linked with cardiac ischemia/reperfusion injury, hypertrophy, and heart failure. While this correlation between ROS and cardiac pathology has been observed in multiple models of heart failure, the independent role of hydrogen peroxide (H2O2) in vitro and in vivo is unclear, owing to a lack of tools for precise manipulation of intracellular redox state. Here we apply a chemogenetic system based on a yeast D-amino acid oxidase to show that chronic generation of H2O2 in the heart induces a dilated cardiomyopathy with significant systolic dysfunction. We anticipate that chemogenetic approaches will enable future studies of in vivo H2O2 signaling not only in the heart, but also in the many other organ systems where the relationship between redox events and physiology remains unclear.

We designed a genetically encoded ratiometric fluorescent probe, SypHer3s, with enhanced brightness and optimized pK, which responds to pH changes in different cellular compartments. SypHer3s was successfully utilized for imaging the pH dynamics in mitochondria of living neurons and in quantitative pH measurement in zebrafish embryos.

Background SypHer is a genetically encoded fluorescent pH-indicator with a ratiometric readout, suitable for measuring fast intracellular pH shifts. However, a relatively low brightness of the indicator limits its use. Methods Here we designed a new version of pH-sensor - SypHer-2, that has up to three times brighter fluorescence signal in cultured mammalian cells compared to the SypHer. Results Using the new indicator we registered activity-associated pH oscillations in neuronal cell culture. We observed prominent temporal neuronal cytoplasm acidification that occurs in parallel with calcium entry. Furthermore, we monitored pH in presynaptic and postsynaptic termini by targeting SypHer-2 directly to these compartments and revealed marked differences in pH dynamics between synaptic boutons and dendritic spines. Finally, we were able to reveal for the first time the intracellular pH drop which occurs within an extended region of the amputated tail of the Xenopus laevis tadpole before it begins to regenerate. Conclusions SypHer2 is suitable for quantitative monitoring of pH in biological systems of different scales, from small cellular subcompartments to animal tissues in vivo. General significance The new pH-sensor will help to investigate pH-dependent processes in both in vitro and in vivo studies.

Thermogenetics is a promising innovative neurostimulation technique, which enables robust activation of neurons using thermosensitive transient receptor potential (TRP) cation channels. Broader application of this approach in neuroscience is, however, hindered by a limited variety of suitable ion channels, and by low spatial and temporal resolution of neuronal activation when TRP channels are activated by ambient temperature variations or chemical agonists. Here, we demonstrate rapid, robust and reproducible repeated activation of snake TRPA1 channels heterologously expressed in non-neuronal cells, mouse neurons and zebrafish neurons in vivo by infrared (IR) laser radiation. A fibre-optic probe that integrates a nitrogen−vacancy (NV) diamond quantum sensor with optical and microwave waveguide delivery enables thermometry with single-cell resolution, allowing neurons to be activated by exceptionally mild heating, thus preventing the damaging effects of excessive heat. The neuronal responses to the activation by IR laser radiation are fully characterized using Ca2+ imaging and electrophysiology, providing, for the first time, a complete framework for a thermogenetic manipulation of individual neurons using IR light.