Chemogenetic Approaches to Studying Cardiovascular Redox Signaling in Vivo
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AbstractOxidant signaling plays many sometimes seemingly opposite roles in both mammalian physiology and disease. Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) generated by cellular respiration and specialized oxidase enzymes have been associated with a diverse set of diseases in multiple organ systems including in the heart, where it is linked with cardiac ischemia/reperfusion injury, hypertrophy and heart failure. However, ROS from these same enzymatic sources have also been found to be necessary for many physiologic signaling processes such as insulin signal transduction and oxygen sensing. The exact role of H2O2 in vitro and in vivo is unclear, owing to a lack of tools for precise manipulation of intracellular redox state. Here we develop a chemogenetic system which allows an experimenter to control H2O2 production in vitro and in vivo through the addition and removal of a D-amino acid substrate. We then applied this system to demonstrate that chronic generation of H2O2 in the heart induces a dilated cardiomyopathy with significant systolic dysfunction. We found that delivery of a fusion protein between the H2O2-sensitive fluorescent biosensor HyPer and a yeast D-amino acid oxidase (DAAO) via an adeno-associated virus type 9 vector achieved robust expression in the hearts of rats. In vitro stimulation of cardiac myocytes from these animals with D-alanine induced a significant transcriptional stress response with increased expression of targets of Nrf2 and NF-kB. In vivo activation of DAAO through the addition of D-alanine to the animals’ drinking water resulted in dephosphorylation of phospholam- ban, decreased expression of alpha myosin heavy chain and severe systolic dysfunction over several weeks. Our findings demonstrate that in vivo H2O2 generation in cardiac myocytes through chemogenetics induces a state of systolic heart failure in the absence of significant fibrotic remodelling. We anticipate that these advances in chemogenetic technology 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, physiology and pathology remains unclear.
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