Pharmacological inhibition of USP14 attenuates LPS-induced septic myocardial injury by rebalancing Bach1/Nrf2 signalling and mitochondrial quality control.

Background

Septic cardiomyopathy is driven by immunoinflammatory activation, oxidative stress, and mitochondrial quality-control failure. However, whether pharmacological targeting of USP14 can attenuate septic myocardial injury through Bach1/Nrf2-associated inflammatory and mitochondrial responses remains unclear.

Methods

HL-1 cardiomyocytes stimulated with lipopolysaccharide and an LPS-induced murine model of septic cardiomyopathy were used in this study. USP14 inhibition was achieved by genetic silencing or pharmacological treatment with IU1. Cardiac function, mitochondrial membrane potential, mitochondrial reactive oxygen species, autophagy-related responses, mitophagy, and related signalling pathways were evaluated using echocardiography, JC-1 staining, flow cytometry, bafilomycin A1-based cellular flux assays, mt-Keima imaging, chloroquine-based assessment of bulk lysosome-dependent autophagic flux in myocardial tissue, co-immunoprecipitation, K48-linked ubiquitination assays, and Western blotting. Rescue experiments were performed to verify the role of Bach1/Nrf2 signalling.

Results

In LPS-stimulated cardiomyocytes, septic stress was associated with Bach1 accumulation, suppression of Nrf2 signalling, reduced mt-Keima-detected mitophagy, mitochondrial depolarization, and oxidative injury. Co-immunoprecipitation detected USP14 in Bach1 immunoprecipitates, while Bach1 ubiquitination analyses showed increased K48-linked ubiquitination of Bach1 after USP14 knockdown. These findings support an association between USP14 and Bach1 and are consistent with, but do not establish, a direct role of USP14 in regulating Bach1 ubiquitination or turnover. Inhibition of USP14 significantly restored Nrf2-related antioxidant signalling, increased mt-Keima-detected mitophagy, reduced mitochondrial superoxide generation, and preserved ATP production and mtDNA copy number in cardiomyocytes. Bach1 overexpression attenuated these protective effects, whereas subsequent Nrf2 overexpression partially rescued the Bach1-mediated attenuation. In septic mice, IU1 treatment improved left ventricular systolic function, reduced myocardial injury and apoptosis, and partially normalized mitochondrial quality-control signalling. CQ-based flux analysis showed significantly greater CQ-induced accumulation of both LC3-II and p62 in IU1-treated mice than in septic mice, supporting enhanced bulk lysosome-dependent autophagic flux in whole myocardial tissue.

Conclusion

USP14 inhibition attenuates LPS-induced inflammatory myocardial injury by rebalancing Bach1/Nrf2 signalling, suppressing oxidative stress, and preserving mitochondrial quality control. These findings suggest that USP14 may serve as a potential pharmacological target for septic cardiomyopathy, although the direct molecular relationship between USP14 and Bach1 requires further clarification.