Introduction: Recently, the p62-Keap1-Nrf2 axis has been identified as a master signaling cascade in the regulation of cellular oxidative stress. Thus, any positive regulators of the p62-Keap1-Nrf2 axis could offer protection against ischemia/reperfusion (I/R)-induced cardiac injury. Our latest data showed that p62 was co-localized with tumor susceptibility gene 101 (Tsg101) in myocytes, and both were simultaneously downregulated in mouse I/R hearts.
Hypothesis: Overexpression of Tsg101 may protect against I/R-caused cardiac injury by activating the p62-Keap1-Nrf2 pathway.
Methods and Results: A transgenic (TG) mouse model with cardiac-specific overexpression of Tsg101 was generated (4-fold). TG and wild-type (WT) mouse hearts underwent global no-flow I/R (45min/1h) with the Langendorff system. TG hearts exhibited improved recovery of contractile performance over the reperfusion period (n=6, p<0.01). This improvement was accompanied by decreased extent of apoptosis (DNA fragmentation, TUNEL-positive nuclei and caspase-3 activity) and a 41% reduction in lactate dehydrogenase release (n=6, p<0.05) in TG hearts compared with WTs. In addition, myocardial infarction size was greatly reduced in TG hearts upon in vivo I/R compared with WTs (n=8, p<0.05). We also generated an inducible cardiac-specific Tsg101-knockdown (KD) mouse model which underwent ex vivo and in vivo I/R, and showed deleterious effects. Mechanistically, we found that protein levels of p62 were increased by 8-fold in TG hearts, together with decreased Keap1 (47%) and increased Nrf2 (3-fold), compared to WTs. Co-immunoprecipitation data showed that Tsg101 was directly bound to p62 in myocytes. We further validated that the p62-Keap1-Nrf2 axis was activated in adenoviral vector (Ad.Tsg101)-infected neonatal rat cardiomyocytes, compared with Ad.GFP-cells. Lastly, overexpression of Tsg101 in cardiomyocytes led to increased cell survival upon H2O2 challenge, which was blocked by either shRNA-mediated knockdown of p62 or addition of alkaloid trigonelline, a potent Nrf2 signal inhibitor, compared with respective controls.
Conclusions: Our data indicate that elevation of Tsg101 could protect against myocardial I/R injury by activating the p62-Keap1-Nrf2 signaling.