Abstract P336: Endogenous Alpha-1a Adrenergic Receptors Promote Cardiomyocyte Survival Through Suppression Of Rip Kinase-mediated Necroptosis

Our previous work has demonstrated essential protective roles for the endogenous cardiomyocyte alpha-1A adrenergic receptor (α1A-AR) subtype in mouse models of heart failure. However, the underlying mechanism of this protective phenotype is unclear. To address this gap in knowledge, we bred a mouse line lacking α1A-ARs on cardiomyocytes by crossing αMHC-cre mice with floxed α1A mice (CMKO= cre+ fl/fl, CMWT= cre- fl/fl), and subjected males to permanent LAD ligation. CMKO mice had increased serum HMGB1 level, larger infarcts and higher mortality. We found that RIP1/3-mediated programmed necrosis (necroptosis), but not apoptosis was exaggerated in CMKO mice 3 days after ligation. We then tested whether RIP1 inhibition with Nec-1s could mitigate this injury. Mice were given Nec-1s (1.65 mg/kg) or vehicle 10 mins prior to LAD ligation, followed by daily IV injection. Nec-1s treatment diminished post-ligation RIP1 (0.62±0.02 vs. 0.78±0.23 A.U., p=NS) and RIP3 expression (0.33±0.1 vs. 0.26±0.10 A.U., p=NS) in CMWT and CMKO mice respectively. Serum level of HMGB1 on D3 was markedly reduced in both CMWT (45.1%) and CMKO (61.1 %) after Nec-1s treatment. There was no difference between Nec-1s treated CMWT and CMKO mice (147±53 vs. 174±37 pg/mL, p=NS), indicating that blocking the RIP kinase pathway abrogates the exaggerated cell death in CMKO mice after ligation. Likewise, Nec-1s-treated CMKO mice had similar infarct areas to CMWT controls (16.2±4.5 vs. 19.9±4.6%, p=NS), further confirming that targeting necroptosis abrogates pathological damage. Collectively these Nec-1s data suggest that RIP-mediated necroptosis may account for larger infarcts in CMKO mice. Interestingly, expression of the apoptosis markers c-caspase-3 and PARP was similar between CMWT and CMKO mice, suggesting that the α1A-AR specifically regulates necroptosis. In sum, our data demonstrate that RIP kinase-mediated necroptosis contributes to susceptibility to injury in mice lacking cardiomyocyte α1A-ARs.

Ferroptosis: A Trigger of Proinflammatory State Progression to Immunogenicity in Necroinflammatory Disease

Until recently, necrosis is generally regarded as traumatic cell death due to mechanical shear stress or other physicochemical factors, while apoptosis is commonly thought to be programmed cell death, which is silent to immunological response. Actually, multiple modalities of cell death are programmed to maintain systematic immunity. Programmed necrosis, such as necrosis, pyroptosis, and ferroptosis, are inherently more immunogenic than apoptosis. Programmed necrosis leads to the release of inflammatory cytokines, defined as danger-associated molecular patterns (DAMPs), resulting in a necroinflammatory response, which can drive the proinflammatory state under certain biological circumstances. Ferroptosis as a newly discovered non-apoptotic form of cell death, is characterized by excessive lipid peroxidation and overload iron, which occurs in cancer, neurodegeneration, immune and inflammatory diseases, as well as ischemia/reperfusion (I/R) injury. It is triggered by a surplus of reactive oxygen species (ROS) induced in an imbalanced redox reaction due to the decrease in glutathione synthesis and inaction of enzyme glutathione peroxidase 4 (GPX4). Ferroptosis is considered as a potential therapeutic and molecular target for the treatment of necroinflammatory disease, and further investigation into the underlying pathophysiological characteristics and molecular mechanisms implicated may lay the foundations for an interventional therapeutic strategy. This review aims to demonstrate the key roles of ferroptosis in the development of necroinflammatory diseases, the major regulatory mechanisms involved, and its potential as a therapeutic target.

Deletion of TLR4 reduces apoptosis and improves histology in a murine kidney transplant model

Acute kidney injury (AKI) after transplantation of human deceased donor kidneys is associated with upregulation of tubular toll like receptor 4 (TLR4), but whether TLR4 is required for AKI is unknown. We hypothesized that TLR4 knockout mice (TLR4KO) subjected to cold ischemia followed by kidney transplant (CI + Txp) would be protected from AKI. C57Bl/6J wild type or TLR4KO kidneys were subjected to CI + Txp into wild type recipients. Tubular cell apoptosis, tubular injury and cast formation were significantly improved in recipients of TLR4KO kidneys. TLR4KO kidneys also demonstrated significantly decreased expression of the effector caspase 8. Brush border injury scores and serum creatinine were not different in recipients of TLR4KO versus wild type kidneys. Phosphorylated RIP3 and MLKL through which TLR4 signals programmed necrosis were expressed in both recipient groups. In addition, TNF-α and TNFR1 expression were significantly increased in recipient serum and TLR4KO kidneys respectively after CI + Txp, suggesting continued activation of programmed necrosis despite TLR4 deletion. Our results suggest that TLR4 deletion decreases apoptosis via inhibition of the death receptor pathway and decreases tubular injury and cast formation.

Neutrophils are important for the development of pro-reparative macrophages after irreversible electroporation of the liver in mice

Irreversible electroporation (IRE) is a non-thermal tissue ablative technology that has emerging applications in surgical oncology and regenerative surgery. To advance its therapeutic usefulness, it is important to understand the mechanisms through which IRE induces cell death and the role of the innate immune system in mediating subsequent regenerative repair. Through intravital imaging of the liver in mice, we show that IRE produces distinctive tissue injury features, including delayed yet robust recruitment of neutrophils, consistent with programmed necrosis. IRE treatment converts the monocyte/macrophage balance from pro-inflammatory to pro-reparative populations, and depletion of neutrophils inhibits this conversion. Reduced generation of pro-reparative Ly6CloF4/80hi macrophages correlates with lower numbers of SOX9+ hepatic progenitor cells in areas of macrophage clusters within the IRE injury zone. Our findings suggest that neutrophils play an important role in promoting the development of pro-reparative Ly6Clo monocytes/macrophages at the site of IRE injury, thus establishing conditions of regenerative repair.

Reactive Oxygen Species Regulate Endoplasmic Reticulum Stress and ER-Mitochondrial Ca2+ Crosstalk to Promote Programmed Necrosis of Rat Nucleus Pulposus Cells under Compression

Programmed necrosis of nucleus pulposus (NP) cells caused by excessive compression is a crucial factor in the etiopathogenesis of intervertebral disc degeneration (IVDD). The endoplasmic reticulum (ER) and mitochondria are crucial regulators of the cell death signaling pathway, and their involvement in IVDD has been reported. However, the specific role of ER stress (ERS) and ER-mitochondria interaction in compression-induced programmed necrosis of NP cells remains unknown. Our studies revealed that compression enhanced ERS and the association between ER and mitochondria in NP cells. Suppression of ERS via 4-phenylbutyrate (4-PBA) or ER-mitochondrial Ca2+ crosstalk by inhibiting the inositol 1,4,5-trisphosphate receptor, glucose-regulated protein 75, voltage-dependent anion-selective channel 1 complex (IP3R–GRP75–VDAC1 complex) protected NP cells against programmed necrosis related to the poly(ADP-ribose) polymerase (PARP) apoptosis-inducing factor (AIF) pathway. Moreover, excessive reactive oxygen species are critical activators of ERS, leading to mitochondrial Ca2+ accumulation and consequent programmed necrosis. These data indicate that ERS and ER-mitochondrial Ca2+ crosstalk may be potential therapeutic targets for the treatment of IVDD-associated disorders. These findings provide new insights into the molecular mechanisms underlying IVDD and may provide novel therapeutic targets.

The molecular mechanisms of MLKL-dependent and MLKL-independent necrosis

Necrosis, a type of unwanted and passive cell demise, usually occurs under the excessive external stress and is considered to be unregulated. However, under some special conditions such as caspase inhibition, necrosis is regulable in a well-orchestrated way. The term ‘regulated necrosis’ has been proposed to describe such programmed necrosis. Recently, several forms of necrosis, including necroptosis, pyroptosis, ferroptosis, parthanatos, oxytosis, NETosis, and Na+/K+-ATPase-mediated necrosis, have been identified, and some crucial regulators governing regulated necrosis have also been discovered. Mixed lineage kinase domain-like pseudokinase (MLKL), a core regulator in necroptosis, acts as an executioner in response to ligands of death receptor family. Its activation requires the receptor-interacting protein kinases, RIP1 and RIP3. However, MLKL is only involved in necroptosis, that is, MLKL is dispensable for necrosis. Therefore, this review is aimed at summarizing the molecular mechanisms of MLKL-dependent and MLKL-independent necrosis.