Chronic GPER1 Activation Protects Against Oxidative Stress-Induced Cardiomyoblast Death via Preservation of Mitochondrial Integrity and Deactivation of Mammalian Sterile-20-Like Kinase/Yes-Associated Protein Pathway.

Introduction: Estrogen (17ß-estradiol, E2) is well-known to induce cardioprotective effects against ischemia/reperfusion (I/R) injury. We recently reported that acute application of E2 at the onset of reperfusion in vivo induces cardioprotective effects against I/R injury via activation of its non-steroidal receptor, G protein-coupled estrogen receptor 1 (GPER1). Here, we investigated the impact and mechanism underlying chronic GPER1 activation in cultured H9c2 rat cardiomyoblasts. Methods: H9c2 rat cardiomyoblasts were cultured and pretreated with the cytotoxic agent H2O2 for 24 h and incubated in the presence of vehicle (control), GPER1 agonists E2 and G1, or GPER1 agonists supplemented with G15 (GPER1 antagonist) for 48 or 96 h. After treatment, cells were collected to measure the rate of cell death and viability using flow cytometry and Calcein AM assay or MTT assay, respectively. The resistance to opening of the mitochondrial permeability transition pore (mPTP), the mitochondrial membrane potential, and ATP production was assessed using fluorescence microscopy, and the mitochondrial structural integrity was observed with electron microscopy. The levels of the phosphorylation of mammalian sterile-20-like kinase (MST1) and yes-associated protein (YAP) were assessed by Western blot analysis in whole-cell lysate, while the expression levels of mitochondrial biogenesis genes, YAP target genes, and proapoptotic genes were measured by qRT-PCR. Results: We found that after H2O2 treatment, chronic E2/G1 treatment decreased cell death effect was associated with the prevention of the S phase of the cell cycle arrest compared to control. In the mitochondria, chronic E2/G1 activation treatment preserved the cristae morphology, and increased resistance to opening of mPTP, but with little change to mitochondrial fusion/fission. Additionally, chronic E2/G1 treatment predominantly reduced phosphorylation of MST1 and YAP, as well as increased MST1 and YAP protein levels. E2 treatment also upregulated the expression levels of TGF-ß and PGC-1α mRNAs and downregulated PUMA and Bim mRNAs. Except for ATP production, all the E2 or G1 effects were prevented by the cotreatment with the GPER1 antagonist, G15. Conclusion: Together, these results indicate that chronic GPER1 activation with its agonists E2 or G1 treatment protects H9c2 cardiomyoblasts against oxidative stress-induced cell death and increases cell viability by preserving mitochondrial structure and function as well as delaying the opening of mPTP. These chronic GPER1 effects are associated with the deactivation of the non-canonical MST1/YAP mechanism that leads to genetic upregulation of cell growth genes (CTGF, CYR61, PGC-1α, and ANKRD1), and downregulation of proapoptotic genes (PUMA and Bim).

Inner mitochondrial membrane protein MPV17 mutant mice display increased myocardial injury after ischemia/reperfusion.

MPV17 is an inner mitochondrial membrane protein whose mutation results in mitochondrial DNA (mtDNA) depletion diseases such as neurohepatopathy. MPV17 is expressed in several organs including the liver and kidneys. Here, we investigated its role and mechanism of action in cardiac ischemia/reperfusion (I/R) injury. Using isolated hearts from wild type and Mpv17 mutant (Mpv17mut) mice, we found that mtDNA levels and normal cardiac function were similar between the groups. Furthermore, reactive oxygen species (ROS) generation, mitochondrial morphology, and calcium levels required to trigger mitochondrial permeability transition pore (mPTP) opening were all similar in normal/non-ischemic animals. However, following I/R, we found that mutant mice had poorer cardiac functional recovery and exhibited more mitochondrial structural damage. We also found that after I/R, Mpv17mut heart mitochondria did not produce more ROS than wild type hearts but that calcium retention capacity was gravely compromised. Using immunoprecipitation and mass spectrometry, we identified ATP synthase, Cyclophilin D, MIC60 and GRP75 as proteins critical to mitochondrial cristae organization and calcium handling that interact with MPV17, and this interaction is reduced by I/R. Together our results suggest that MPV17 has a protective function in the heart and is necessary for recovery following insults to the heart.

Liproxstatin-1 protects the mouse myocardium against ischemia/reperfusion injury by decreasing VDAC1 levels and restoring GPX4 levels.

Ferroptosis is a distinct iron-dependent mechanism of regulated cell death recognized in cancer and ischemia/reperfusion (I/R) injury of different organs. It has been reported that molecules such as liproxstatin-1 (Lip-1) inhibit ferroptosis and promote cell survival however, the mechanisms underlying this action are not clearly understood. We investigated the role and mechanism of Lip-1 in reducing cell death in the ischemic myocardium. Using an I/R model of isolated perfused mice hearts in which Lip-1 was given at the onset of reperfusion, we found that Lip-1 protects the heart by reducing myocardial infarct sizes and maintaining mitochondrial structural integrity and function. Further investigation revealed that Lip-1-induced cardioprotection is mediated by a reduction of VDAC1 levels and oligomerization, but not VDAC2/3. Lip-1 treatment also decreased mitochondrial reactive oxygen species production and rescued the reduction of the antioxidant GPX4 caused by I/R stress. Meanwhile, mitochondrial Ca2+ retention capacity needed to induce mitochondrial permeability transition pore opening did not change with Lip-1 treatment. Thus, we report that Lip-1 induces cardioprotective effects against I/R injury by reducing VDAC1 levels and restoring GPX4 levels.

Mitochondrial inner membrane protein, Mic60/mitofilin in mammalian organ protection.

The identification of the mitochondrial contact site and cristae organizing system (MICOS) in the inner mitochondrial membrane shed light on the intricate components necessary for mitochondria to form their signature cristae in which many protein complexes including the electron transport chain are localized. Mic60/mitofilin has been described as the core component for the assembly and maintenance of MICOS, thus controlling cristae morphology, protein transport, mitochondrial DNA transcription, as well as connecting the inner and outer mitochondrial membranes. Although Mic60 homologs are present in many species, mammalian Mic60 is only recently gaining attention as a critical player in several organ systems and diseases with mitochondrial-defect origins. In this review, we summarize what is currently known about the ever-expanding role of Mic60 in mammals, and highlight some new studies pushing the field of mitochondrial cristae organization towards potentially new and exciting therapies targeting this protein.

Mitochondrial inner membrane protein (mitofilin) knockdown induces cell death by apoptosis via an AIF-PARP-dependent mechanism and cell cycle arrest.

Mitofilin is an inner membrane protein that has been defined as a mitochondria-shaping protein in controlling and maintaining mitochondrial cristae structure and remodeling. We determined the role of mitofilin in cell survival by investigating the mechanism underlying mitofilin knockdown-induced cell death by apoptosis. Cultured H9c2 myoblasts and HEK 293 cells were treated with mitofilin siRNA or scrambled siRNA for 24 h. Cell death (apoptosis), caspase 3 activity and cell cycle phases were assessed by flow cytometry, while cytochrome c release and intracellular ATP production were measured by ELISA. Mitofilin, apoptosis-inducing factor (AIF) and poly(ADP-ribose) polymerase (PARP) expression were measured by Western blot analysis and calpain activity was assessed using a calpain activity kit. Mitochondrial images were taken using electron microscopy. We found that mitofilin knockdown increases apoptosis mainly via activation of the AIF-PARP pathway leading to nuclear fragmentation that is correlated with S phase arrest of the cell cycle. Knockdown of mitofilin also led to mitochondrial swelling and damage of cristae that is associated with the increase in reactive oxygen species production and mitochondrial calpain activity, as well as a marked decrease in intracellular ATP production and mitochondrial membrane potential. Together, these results indicate that mitofilin knockdown by siRNA increases calpain activity that presumably leads to mitochondrial structural degradation resulting in a critical reduction of mitochondrial function that is responsible for the increase in cell death by apoptosis via an AIF-PARP mechanism and associated with nuclear fragmentation, and S phase arrest of the cell cycle.

TrpA1 activation in peripheral sensory neurons underlies the ionic basis of pain hypersensitivity in response to vinca alkaloids.

Chemotherapy induced peripheral neuropathy (CIPN), a side effect of many anti-cancer drugs including the vinca alkaloids, is characterized by a severe pain syndrome that compromises treatment in many patients. Currently there are no effective treatments for this pain syndrome except for the reduction of anti-cancer drug dose. Existing data supports the model that the pain associated with CIPN is the result of anti-cancer drugs augmenting the function of the peripheral sensory nociceptors but the cellular mechanisms underlying the effects of anti-cancer drugs on sensory neuron function are not well described. Studies from animal models have suggested a number of disease etiologies including mitotoxicity, axonal degeneration, immune signaling, and reduced sensory innervations but these outcomes are the result of prolonged treatment paradigms and do not necessarily represent the early formative events associated with CIPN. Here we show that acute exposure to vinca alkaloids results in an immediate pain syndrome in both flies and mice. Furthermore, we demonstrate that exposure of isolated sensory neurons to vinca alkaloids results in the generation of an inward sodium current capable of depolarizing these neurons to threshold resulting in neuronal firing. These neuronal effects of vinca alkaloids require the transient receptor potential ankyrin-1 (TrpA1) channel, and the hypersensitization to painful stimuli in response to the acute exposure to vinca alkaloids is reduced in TrpA1 mutant flies and mice. These findings demonstrate the direct excitation of sensory neurons by CIPN-causing chemotherapy drugs, and identify TrpA1 as an important target during the pathogenesis of CIPN.

Activation of G protein-coupled oestrogen receptor 1 at the onset of reperfusion protects the myocardium against ischemia/reperfusion injury by reducing mitochondrial dysfunction and mitophagy.

BACKGROUND AND PURPOSE: Recent evidence indicates that GPER (G protein-coupled oestrogen receptor 1) mediates acute pre-ischaemic oestrogen-induced protection of the myocardium from ischaemia/reperfusion injury via a signalling cascade that includes PKC translocation, ERK1/2/ GSK-3ß phosphorylation and inhibition of the mitochondrial permeability transition pore (mPTP) opening. Here, we investigated the impact and mechanism involved in post-ischaemic GPER activation in ischaemia/reperfusion injury. We determined whether GPER activation at the onset of reperfusion confers cardioprotective effects by protecting against mitochondrial impairment and mitophagy. EXPERIMENTAL APPROACH: In vivo rat hearts were subjected to ischaemia followed by reperfusion with oestrogen (17ß-oestradiol, E2), E2 + G15, a GPER antagonist, or vehicle. Myocardial infarct size, the threshold for the opening of mPTP, mitophagy, mitochondrial membrane potential, ROS production, proteins ubiquitinated including cyclophilin D, and phosphorylation levels of ERK and GSK-3ß were measured. RESULTS: We found that post-ischaemic E2 administration to both male and female ovariectomized-rats reduced myocardial infarct size. Post-ischaemic E2 administration preserved mitochondrial structural integrity and this was associated with a decrease in ROS production and increased mitochondrial membrane potential, as well as an increase in the mitochondrial Ca2+ load required to induce mPTP opening via activation of the MEK/ERK/GSK-3ß axis. Moreover, E2 reduced mitophagy via the PINK1/Parkin pathway involving LC3I, LC3II and p62 proteins. All these post-ischaemic effects of E2 were abolished by G15 suggesting a GPER-dependent mechanism. CONCLUSION: These results indicate that post-ischaemic GPER activation induces cardioprotective effects against ischaemia/reperfusion injury in males and females by protecting mitochondrial structural integrity and function and reducing mitophagy.

Critical role of mitochondrial ROS is dependent on their site of production on the electron transport chain in ischemic heart.

Reactive oxygen species (ROS) generation has been implicated in many pathologies including ischemia/reperfusion (I/R) injury. This led to multiple studies on antioxidant therapies to treat cardiovascular diseases but paradoxically, results have so far been mixed as ROS production can be beneficial as a signaling mechanism and in cardiac protection via preconditioning interventions. We investigated whether the differential impact of increased ROS in injury as well as in protection could be explained by their site of production on the mitochondrial electron transport chain. Using amplex red to measure ROS production, we found that mitochondria isolated from hearts after I/R produced more ROS than non-ischemic when complex I substrate (glutamate/malate) was used. Interestingly, the substrates of complex II (succinate) and ubiquinone (sn-glycerol 3-phosphate, G3P) produced less ROS in mitochondria from I/R hearts compared to normal healthy hearts. The inhibitors of complex I (rotenone) and complex III (antimycin A) increased ROS production when glutamate/malate and G3P were used; in contrast, they reduced ROS production when the complex II substrate was used. Mitochondrial calcium retention capacity required to induce mitochondrial permeability transition pore (mPTP) opening was measured using calcium green fluorescence and was found to be higher when mitochondria were treated with G3P and succinate compared to glutamate/malate. Furthermore, Langendorff hearts treated with glutamate/malate exhibited reduced cardiac functional recovery and increased myocardial infarct size compared to hearts treated with G3P. Thus, ROS production by the stimulated respiratory chain complexes I and III has opposite roles: cardio-deleterious when produced in complex I and cardio-protective when produced in complex III. The mechanism of these ROS involves the inhibition of the mPTP opening, a key event in cell death following ischemia/reperfusion injury.