Role of hexokinase 3 in macrophage polarization and cardiac remodeling after myocardial infraction. - Project Summary Myocardial infarction (MI) is a leading cause of death, and macrophages play a critical role in the remodeling of the heart after MI. Hexokinases (HKs) catalyze glucose phosphorylation, and one HK isoform (HK3) is mostly expressed in myeloid cells. We have generated global and macrophage-specific (MS)-Hk3-/- mice and have shown that deletion of Hk3 leads to protection against cardiac ischemic injury and enhances polarization to the repair M2 subtype with unaltered M1/classical activation. Bone marrow derived macrophages (BMDMs) and circulating monocytes from mice with Hk3 deletion display increased glutathione levels and NRF2 activity; also, acute inactivation of HK3 leads to increased reactive oxygen species (ROS). These results suggest that HK3 plays a major role in macrophage polarization and that its deletion increases macrophage repair phenotype, NRF2 activation and glutathione production, and protects against ischemic injury. In this proposal, we will address the fundamental gap in knowledge of the role of macrophages and their metabolic processes in cardiac remodeling after ischemia/reperfusion (I/R). Our central hypothesis is that HK3 plays a major role in macrophage polarization, such that its deletion results in polarization towards the repair phenotype and an improvement in cardiac remodeling after I/R. We also propose that the mechanism for the M2 polarization with Hk3 deletion is through NRF2 activation and glutathione production. In Aim 1, we will determine whether HK3 restrains NRF2, so that when it is deleted, NRF2 gets activated leading to glutathione synthesis. We also hypothesize that the mechanism for NRF2 activation is through an initial increase in ROS production. We will obtain cells at early and late timepoints after Hk3 deletion by crossing Hk3f/f mice with CX3CR1-CRE-ERT2 and assess: 1) ROS levels, 2) NADPH levels, 3) NRF2 activation, 4) glutathione production, and 5) glucose carbon tracing. We will also assess whether antioxidants reverse NRF2 activation and glutathione production. In Aim 2, we will determine whether deletion of Hk3 drives macrophage polarization through the transcriptional or metabolic effects of NRF2. We will first cross MS-Hk3-/- with Nrf2-/- mice and assess M2 polarization. We will also test whether Hk3 deletion drives macrophage polarization through direct transcriptional activation of M2 genes by performing RNA-Seq and ChIP-Seq. Finally, we will test whether the effects of NRF2 on glutathione or its antioxidant effects mediate the macrophage polarization induced by Hk3 deletion by measuring polarization of macrophages in MS-Keap1-/- mice (constitutively active NRF2) or with buthionine sulfoximine (BSO, an inhibitor of glutathione production) or ROS inducers. In Aim 3, we will determine whether Hk3 deletion protects against I/R through improved cardiac remodeling and will identify the cell populations that exert this protective effect. We will subject Hk3-/-, MS-Hk3-/, and mice with neutrophil-specific Hk3 deletion to I/R, followed by measurement of cardiac function and remodeling markers. We will also assess the role of cardiac-resident and tissue macrophages that originate from circulating monocytes in this process.
