Exosomes in Cardiac Preconditioning with Isoflurane and Hypoxia

Objective: Cardiac preconditioning with brief cycles of hypoxia or volatile anesthetics like isoflurane leads to a significant protection from subsequent ischemia/reperfusion injury. The crosstalk between different cardiac cell types (cardiomyocytes, fibroblasts, endothelial cells) has come into focus of current research. One possibility is the intercellular exchange of cardioprotective mediators like heat shock proteins (HSP). In this context exosomes, which are small membrane vesicles that are actively secreted, have emerged as possible communication mediators. Recent publications indicate, that exosomes might be involved in hypoxic preconditioning, but mechanistical data still lacks. In addition it remains to be investigated whether other preconditioning stimuli, like volatile anaesthetics also trigger the release of vesicles and if these exosomes differ in their protein composition.

Methods: Primary cardiac fibroblasts were isolated from neonatal rats via trypsin digestion and subsequent preplating. Cells were exposed to different preconditioning stimuli like isoflurane (1.5 vol%, 4h) or hypoxia (< 1% O₂, 1h) and supernatants were collected after 16h. Exosomes were isolated via differential centrifugation and further purified by ultracentrifugation on a 30% sucrose/D₂O cushion. Isolated exosomes were quantified and characterized via different methods: 1) protein quantification using micro BCA assay, 2) western blot analysis with detection of the exosomal marker proteins CD63 and HSP/HSC70, 3) exosome ELISA which quantifies CD63-positive vesicles and 4) dynamic light scattering (DLS).

Results: Exosomes were successfully isolated from supernatants of preconditioned fibroblasts by sequential centrifugation. Even without stimulus, fibroblasts constantly secrete low amounts of exosomes, but preconditioning results in a 3–5 fold increased release of vesicles. Preconditioning with isoflurane and hypoxia triggered the specific release of CD63+ vesicles. Interestingly, HSP/HSC70 was only present in vesicles after isoflurane treatment, which implies that different preconditioning stimuli indeed trigger the release of vesicles with varying protein compositions.

Conclusion: We were able to show, that primary cardiac fibroblasts actively secrete exosomes after a preconditioning stimulus. The protein composition of theses vesicles differs, depending on the stimulus used and might indicate differential roles during preconditioning.