Inhibition of the mPTP and Lipid Peroxidation Is Additively Protective Against I/R Injury.

BACKGROUND: During myocardial ischemia/reperfusion (I/R) injury, high levels of matrix Ca2+ and reactive oxygen species (ROS) induce the opening of the mitochondrial permeability transition pore (mPTP), which causes mitochondrial dysfunction and ultimately necrotic death. However, the mechanisms of how these triggers individually or cooperatively open the pore have yet to be determined. METHODS: Here, we use a combination of isolated mitochondrial assays and in vivo I/R surgery in mice. We challenged isolated liver and heart mitochondria with Ca2+, ROS, and Fe2+ to induce mitochondrial swelling. Using inhibitors of the mPTP (cyclosporine A or ADP) lipid peroxidation (ferrostatin-1, MitoQ), we determined how the triggers elicit mitochondrial damage. Additionally, we used the combination of inhibitors during I/R injury in mice to determine if dual inhibition of these pathways is additivity protective. RESULTS: In the absence of Ca2+, we determined that ROS fails to trigger mPTP opening. Instead, high levels of ROS induce mitochondrial dysfunction and rupture independently of the mPTP through lipid peroxidation. As expected, Ca2+ in the absence of ROS induces mPTP-dependent mitochondrial swelling. Subtoxic levels of ROS and Ca2+ synergize to induce mPTP opening. Furthermore, this synergistic form of Ca2+- and ROS-induced mPTP opening persists in the absence of CypD (cyclophilin D), suggesting the existence of a CypD-independent mechanism for ROS sensitization of the mPTP. These ex vivo findings suggest that mitochondrial dysfunction may be achieved by multiple means during I/R injury. We determined that dual inhibition of the mPTP and lipid peroxidation is significantly more protective against I/R injury than individually targeting either pathway alone. CONCLUSIONS: In the present study, we have investigated the relationship between Ca2+ and ROS, and how they individually or synergistically induce mitochondrial swelling. Our findings suggest that Ca2+ mediates mitochondrial damage through the opening of the mPTP, although ROS mediates its damaging effects through lipid peroxidation. However, subtoxic levels both Ca2+ and ROS can induce mPTP-mediated mitochondrial damage. Targeting both of these triggers to preserve mitochondria viability unveils a highly effective therapeutic approach for mitigating I/R injury.

A High-Protein Diet Promotes Atrial Arrhythmogenesis via Absent-in-Melanoma 2 Inflammasome.

High-protein diets (HPDs) offer health benefits, such as weight management and improved metabolic profiles. The effects of HPD on cardiac arrhythmogenesis remain unclear. Atrial fibrillation (AF), the most common arrhythmia, is associated with inflammasome activation. The role of the Absent-in-Melanoma 2 (AIM2) inflammasome in AF pathogenesis remains unexplored. In this study, we discovered that HPD increased susceptibility to AF. To demonstrate the involvement of AIM2 signaling in the pathogenesis of HPD-induced AF, wildtype (WT) and Aim2-/- mice were fed normal-chow (NC) and HPD, respectively. Four weeks later, inflammasome activity was upregulated in the atria of WT-HPD mice, but not in the Aim2-/--HPD mice. The increased AF vulnerability in WT-HPD mice was associated with abnormal sarcoplasmic reticulum (SR) Ca2+-release events in atrial myocytes. HPD increased the cytoplasmic double-strand (ds) DNA level, causing AIM2 activation. Genetic inhibition of AIM2 in Aim2-/- mice reduced susceptibility to AF, cytoplasmic dsDNA level, mitochondrial ROS production, and abnormal SR Ca2+-release in atrial myocytes. These data suggest that HPD creates a substrate conducive to AF development by activating the AIM2-inflammasome, which is associated with mitochondrial oxidative stress along with proarrhythmic SR Ca2+-release. Our data imply that targeting the AIM2 inflammasome might constitute a novel anti-AF strategy in certain patient subpopulations.

Caspase-2 is essential for proliferation and self-renewal of nucleophosmin-mutated acute myeloid leukemia.

Mutation in nucleophosmin (NPM1) causes relocalization of this normally nucleolar protein to the cytoplasm ( NPM1c+ ). Despite NPM1 mutation being the most common driver mutation in cytogenetically normal adult acute myeloid leukemia (AML), the mechanisms of NPM1c+-induced leukemogenesis remain unclear. Caspase-2 is a pro-apoptotic protein activated by NPM1 in the nucleolus. Here, we show that caspase-2 is also activated by NPM1c+ in the cytoplasm, and DNA damage-induced apoptosis is caspase-2-dependent in NPM1c+ AML but not in NPM1wt cells. Strikingly, in NPM1c+ cells, loss of caspase-2 results in profound cell cycle arrest, differentiation, and down-regulation of stem cell pathways that regulate pluripotency including impairment in the AKT/mTORC1 and Wnt signaling pathways. In contrast, there were minimal differences in proliferation, differentiation, or the transcriptional profile of NPM1wt cells with and without caspase-2. Together, these results show that caspase-2 is essential for proliferation and self-renewal of AML cells that have mutated NPM1. This study demonstrates that caspase-2 is a major effector of NPM1c+ function and may even be a druggable target to treat NPM1c+ AML and prevent relapse.

Mitochondrial permeability transition pore-dependent necrosis.

Mitochondrial permeability transition pore (mPTP)-dependent cell death is a form of necrotic cell death that is driven by mitochondrial dysfunction by the opening of the mPTP and is triggered by increases in matrix levels of Ca2+ and reactive oxygen species. This form of cell death has been implicated in ischemic injuries of the heart and brain as well as numerous degenerative diseases in the brain and skeletal muscle. This review focuses on the molecular triggers and regulators of mPTP-dependent necrosis in the context of myocardial ischemia reperfusion injury. Research over the past 50 years has led to the identity of regulators and putative pore-forming components of the mPTP. Finally, downstream consequences of activation of the mPTP as well as ongoing questions and areas of research are discussed. These questions pose a particular interest as targeting the mPTP could potentially represent an efficacious therapeutic strategy to reduce infarct size following an ischemic event.

Inhibition of the Anti-Apoptotic Bcl-2 Family by BH3 Mimetics Sensitize the Mitochondrial Permeability Transition Pore Through Bax and Bak.

Mitochondrial permeability transition pore (MPTP)-dependent necrosis contributes to numerous pathologies in the heart, brain, and skeletal muscle. The MPTP is a non-selective pore in the inner mitochondrial membrane that is triggered by high levels of matrix Ca2+, and sustained opening leads to mitochondrial dysfunction. Although the MPTP is defined by an increase in inner mitochondrial membrane permeability, the expression of pro-apoptotic Bcl-2 family members, Bax and Bak localization to the outer mitochondrial membrane is required for MPTP-dependent mitochondrial dysfunction and subsequent necrotic cell death. Contrary to the role of Bax and Bak in apoptosis, which is dependent on their oligomerization, MPTP-dependent necrosis does not require oligomerization as monomeric/inactive forms of Bax and Bak can facilitate mitochondrial dysfunction. However, the relationship between Bax and Bak activation/oligomerization and MPTP sensitization remains to be explored. Here, we use a combination of in vitro and ex vivo approaches to determine the role of the anti-apoptotic Bcl-2 family members, which regulate Bax/Bak activity, in necrotic cell death and MPTP sensitivity. To study the role of each predominantly expressed anti-apoptotic Bcl-2 family member (i.e., Mcl-1, Bcl-2, and Bcl-xL) in MPTP regulation, we utilize various BH3 mimetics that specifically bind to and inhibit each. We determined that the inhibition of each anti-apoptotic Bcl-2 family member lowers mitochondrial calcium retention capacity and sensitizes MPTP opening. Furthermore, the inhibition of each Bcl-2 family member exacerbates both apoptotic and necrotic cell death in vitro in a Bax/Bak-dependent manner. Our findings suggests that mitochondrial Ca2+ retention capacity and MPTP sensitivity is influenced by Bax/Bak activation/oligomerization on the outer mitochondrial membrane, providing further evidence of the crosstalk between the apoptotic and necrotic cell death pathways.

RELA is sufficient to mediate interleukin-1 repression of androgen receptor expression and activity in an LNCaP disease progression model.

BACKGROUND: The androgen receptor (AR) nuclear transcription factor is a therapeutic target for prostate cancer (PCa). Unfortunately, patients can develop resistance to AR-targeted therapies and progress to lethal disease, underscoring the importance of understanding the molecular mechanisms that underlie treatment resistance. Inflammation is implicated in PCa initiation and progression and we have previously reported that the inflammatory cytokine, interleukin-1 (IL-1), represses AR messenger RNA (mRNA) levels and activity in AR-positive (AR+ ) PCa cell lines concomitant with the upregulation of prosurvival biomolecules. Thus, we contend that IL-1 can select for AR-independent, treatment-resistant PCa cells. METHODS: To begin to explore how IL-1 signaling leads to the repression of AR mRNA levels, we performed comprehensive pathway analysis on our RNA sequencing data from IL-1-treated LNCaP PCa cells. Our pathway analysis predicted nuclear factor kappa B (NF-κB) p65 subunit (RELA), a canonical IL-1 signal transducer, to be significantly active and potentially regulate many genes, including AR. We used small interfering RNA (siRNA) to silence the NF-κB family of transcription factor subunits, RELA, RELB, c-REL, NFKB1, or NFKB2, in IL-1-treated LNCaP, C4-2, and C4-2B PCa cell lines. C4-2 and C4-2B cell lines are castration-resistant LNCaP sublines and represent progression toward metastatic PCa disease, and we have previously shown that IL-1 represses AR mRNA levels in C4-2 and C4-2B cells. RESULTS: siRNA revealed that RELA alone is sufficient to mediate IL-1 repression of AR mRNA and AR activity. Intriguingly, while LNCaP cells are more sensitive to IL-1-mediated repression of AR than C4-2 and C4-2B cells, RELA siRNA led to a more striking derepression of AR mRNA levels and AR activity in C4-2 and C4-2B cells than in LNCaP cells. CONCLUSIONS: These data indicate that there are RELA-independent mechanisms that regulate IL-1-mediated AR repression in LNCaP cells and suggest that the switch to RELA-dependent IL-1 repression of AR in C4-2 and C4-2B cells reflects changes in epigenetic and transcriptional programs that evolve during PCa disease progression.