Heart failure (HF) is a major health epidemic in the developed countries; however, the underlying molecular mechanisms for this disorder are not well characterized. Sirtuins (SIRT) are proteins that exhibit deacetylation or ADP-ribosyltransferase activity and regulate a wide range of cellular processes. There are seven SIRTs in mammalian cells that reside in the nucleus, mitochondria or cytoplasm, and modify certain proteins in their respective cellular compartments. Nuclear and mitochondrial SIRTs are known to contribute to cardiac protection, but the role of SIRT2 (the only sirtuin that resides predominantly in the cytoplasm) in the heart is not known. We are addressing this fundamental gap in knowledge, and our strong preliminary data indicate that the levels of SIRT2 are increased in failing human and mouse hearts. Furthermore, the hearts of mice with global deletion of Sirt2 (Sirt2-/-) display improved cardiac function after ischemia-reperfusion (I/R) and pressure overload (PO). Thus, SIRT2 may have detrimental effects in the heart under stress conditions, which makes this protein a unique member of the SIRT family. Furthermore, our mechanistic studies suggest that SIRT2 modulates cellular levels and activity of nuclear factor (erythroid-derived 2)-like 2 (NRF2), a transcription factor that induces the expression of antioxidant proteins. Finally, using mass spectrometry and site-directed mutagenesis, we have identified specific lysine residues in NRF2 that are targeted by SIRT2. The central hypothesis of this proposal is that SIRT2 exerts detrimental effects in the heart through deacetylation of NRF2, resulting in a reduction in its protein levels and transcriptional activity, thereby decreasing the expression of antioxidant proteins.
In Aim 1, we will determine whether SIRT2 exerts deleterious effects in the heart in response to injury, and whether the protection noted in Sirt2-/- mice is due to systemic or cardiac-specific deletion of Sirt2. We will subject cardiac specific Sirt2 knockout (cs-Sirt2-/-) mice and mice with overexpression of SIRT2 to PO and will assess cardiac function, as well as reactive oxygen species (ROS) levels using novel fluorescence markers.
In Aim 2, we will study whether SIRT2 regulates NRF2 abundance and gene transcriptional activity by deacetylating specific lysine residues within its DNA-binding and ubiquitination domains. We will assess total and nuclear NRF2 levels, and its ubiquitination and transcriptional activity in the hearts of cs-Sirt2-/- mice at baseline and after PO and in mouse embryonic fibroblasts (MEFs) with Sirt2 knockdown. We will also measure NRF2 total and nuclear levels and transcriptional activity with overexpression of NRF2 mutant constructs with mutation of SIRT2-targetd lysines to acetylated- or deacetylated-mimetic residues.
In Aim 3, we will determine whether the deleterious effects of SIRT2 in response to injury are mediated through NRF2. cs-Sirt2-/- and cs- Sirt2-/-/Nrf2-/- mice will be subjected to PO, followed by assessment of cardiac function and ROS levels. We will also express NRF2 constructs with mutations of SIRT2 deacetylation sites in cardiomyocytes and assess cell death and ROS production.
Heart failure is highly prevalent in developed countries, but the underlying mechanism for this disorder is not well characterized. Sirtuin (SIRT) family of proteins modify other proteins within the cell by removing acetyl groups and altering their function. One member of this family, SIRT2, has not been studied in the heart, and in this proposal, we will assess its role in cardiac response to pressure overload, through its regulation of NRF2 protein activity.
| Sato, Tatsuya; Chang, Hsiang-Chun; Bayeva, Marina et al. (2018) mRNA-binding protein tristetraprolin is essential for cardiac response to iron deficiency by regulating mitochondrial function. Proc Natl Acad Sci U S A 115:E6291-E6300 |
| De Jesus, Adam; Chang, Hsiang-Chun; Ardehali, Hossein (2018) Metabolic Suppression of HIF-1? Contributes to Susceptibility of Ischemic Injury in Diabetic Hearts. JACC Basic Transl Sci 3:499-502 |
| Yang, Xiaoyan; Park, Seong-Hoon; Chang, Hsiang-Chun et al. (2017) Sirtuin 2 regulates cellular iron homeostasis via deacetylation of transcription factor NRF2. J Clin Invest 127:1505-1516 |
