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Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions
Published on: August 2, 2018
Sequestosome 1 (p62) mitigates hypoxia-induced cardiac dysfunction by stabilizing hypoxia-inducible factor 1α and
Rajeshwary Ghosh1,2, Amir Nima Fatahian1, Omid M T Rouzbehani1
1Department of Nutrition and Integrative Physiology, College of Health, University of Utah, Salt Lake City, UT 84112, USA.
Insights
Cardiac sequestosome 1 (p62) protein protects against heart damage caused by low oxygen. Loss of p62 impairs key signaling pathways, leading to heart dysfunction and cell death.
Area of Science:
- Cardiovascular Biology
- Molecular Medicine
- Cellular Signaling
Background:
- Ischaemic heart disease (IHD) is a major cause of heart failure, with hypoxia exacerbating cardiac damage.
- Sequestosome 1 (p62) is an adaptor protein highly expressed in cardiomyocytes, but its role in cardiac physiology remains unclear.
- Understanding p62's function is crucial for developing strategies against IHD-induced cardiac pathology.
Purpose of the Study:
- To investigate the role of cardiomyocyte-specific p62 in cardiac response to hypoxia.
- To determine if p62 deficiency impairs hypoxia-inducible factor 1α (Hif-1α) and nuclear factor erythroid 2-related factor 2 (Nrf2) signaling.
- To elucidate the mechanisms by which p62 influences cardiac function under hypoxic stress.
Main Methods:
- Generated adult mice with germline and inducible cardiomyocyte-specific p62 deletion.
- Performed transcriptomic analyses to assess gene expression changes in p62-deficient hearts.
- Utilized H9c2 cardiomyoblast cell line to investigate molecular mechanisms of p62, Hif-1α, and Nrf2 interaction under hypoxia.
- Assessed protein levels, nuclear translocation, ubiquitination, and transcriptional activity of Nrf2.
Main Results:
- Cardiomyocyte p62 deletion led to cardiac dysfunction, oxidative stress, and cell death under hypoxic conditions.
- p62 deficiency impaired both Hif-1α and Nrf2 transcriptional activity in the heart.
- Loss of p62 in cardiomyoblasts reduced Hif-1α and Nrf2 protein levels and Nrf2 nuclear translocation.
- p62 absence increased Nrf2 ubiquitination and proteasomal degradation via Cullin 3, while p62 gain stabilized Nrf2.
Conclusions:
- Cardiac p62 plays a critical cardioprotective role against hypoxia-induced cardiac dysfunction.
- p62 stabilizes Hif-1α and Nrf2, preserving their transcriptional activity under hypoxic stress.
- Targeting p62 may offer a therapeutic strategy for mitigating heart failure associated with IHD and hypoxia.
Aims:
Heart failure due to ischaemic heart disease (IHD) is a leading cause of mortality worldwide. A major contributing factor to IHD-induced cardiac damage is hypoxia. Sequestosome 1 (p62) is a multi-functional adaptor protein with pleiotropic roles in autophagy, proteostasis, inflammation, and cancer. Despite abundant expression in cardiomyocytes, the role of p62 in cardiac physiology is not well understood. We hypothesized that cardiomyocyte-specific p62 deletion evokes hypoxia-induced cardiac pathology by impairing hypoxia-inducible factor 1α (Hif-1α) and nuclear factor erythroid 2-related factor 2 (Nrf2) signalling.
Methods And Results:
Adult mice with germline deletion of cardiomyocyte p62 exhibited mild cardiac dysfunction under normoxic conditions. Transcriptomic analyses revealed a selective impairment in Nrf2 target genes in the hearts from these mice. Demonstrating the functional importance of this adaptor protein, adult mice with inducible depletion of cardiomyocyte p62 displayed hypoxia-induced contractile dysfunction, oxidative stress, and cell death. Mechanistically, p62-depleted hearts exhibit impaired Hif-1α and Nrf2 transcriptional activity. Because findings from these two murine models suggested a cardioprotective role for p62, mechanisms were evaluated using H9c2 cardiomyoblasts. Loss of p62 in H9c2 cells exposed to hypoxia reduced Hif-1α and Nrf2 protein levels. Further, the lack of p62 decreased Nrf2 protein expression, nuclear translocation, and transcriptional activity. Repressed Nrf2 activity associated with heightened Nrf2-Keap1 co-localization in p62-deficient cells, which was concurrent with increased Nrf2 ubiquitination facilitated by the E3 ligase Cullin 3, followed by proteasomal-mediated degradation. Substantiating our results, a gain of p62 in H9c2 cells stabilized Nrf2 and increased the transcriptional activity of Nrf2 downstream targets.
Conclusion:
Cardiac p62 mitigates hypoxia-induced cardiac dysfunction by stabilizing Hif-1α and Nrf2.
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