Unique amalgamation of primary and secondary structural elements transform peptaibols into potent bioactive cell-penetrating peptides.

Mass-spectrometry-based metabolomics and molecular phylogeny data were used to identify a metabolically prolific strain of Tolypocladium that was obtained from a deep-water Great Lakes sediment sample. An investigation of the isolate's secondary metabolome resulted in the purification of a 22-mer peptaibol, gichigamin A (1). This peptidic natural product exhibited an amino acid sequence including several ß-alanines that occurred in a repeating ααß motif, causing the compound to adopt a unique right-handed 311 helical structure. The unusual secondary structure of 1 was confirmed by spectroscopic approaches including solution NMR, electronic circular dichroism (ECD), and single-crystal X-ray diffraction analyses. Artificial and cell-based membrane permeability assays provided evidence that the unusual combination of structural features in gichigamins conferred on them an ability to penetrate the outer membranes of mammalian cells. Compound 1 exhibited potent in vitro cytotoxicity (GI50 0.55 ± 0.04 µM) and in vivo antitumor effects in a MIA PaCa-2 xenograft mouse model. While the primary mechanism of cytotoxicity for 1 was consistent with ion leakage, we found that it was also able to directly depolarize mitochondria. Semisynthetic modification of 1 provided several analogs, including a C-terminus-linked coumarin derivative (22) that exhibited appreciably increased potency (GI50 5.4 ± 0.1 nM), but lacked ion leakage capabilities associated with a majority of naturally occurring peptaibols such as alamethicin. Compound 22 was found to enter intact cells and induced cell death in a process that was preceded by mitochondrial depolarization.

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.

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 (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.

MitoBK(Ca) is encoded by the Kcnma1 gene, and a splicing sequence defines its mitochondrial location.

The large-conductance Ca(2+)- and voltage-activated K(+) channel (BK(Ca), MaxiK), which is encoded by the Kcnma1 gene, is generally expressed at the plasma membrane of excitable and nonexcitable cells. However, in adult cardiomyocytes, a BK(Ca)-like channel activity has been reported in the mitochondria but not at the plasma membrane. The putative opening of this channel with the BK(Ca) agonist, NS1619, protects the heart from ischemic insult. However, the molecular origin of mitochondrial BK(Ca) (mitoBK(Ca)) is unknown because its linkage to Kcnma1 has been questioned on biochemical and molecular grounds. Here, we unequivocally demonstrate that the molecular correlate of mitoBK(Ca) is the Kcnma1 gene, which produces a protein that migrates at ∼140 kDa and arranges in clusters of ∼50 nm in purified mitochondria. Physiological experiments further support the origin of mitoBK(Ca) as a Kcnma1 product because NS1619-mediated cardioprotection was absent in Kcnma1 knockout mice. Finally, BKCa transcript analysis and expression in adult cardiomyocytes led to the discovery of a 50-aa C-terminal splice insert as essential for the mitochondrial targeting of mitoBK(Ca).

Mitofilin Heterozygote Mice Display an Increase in Myocardial Injury and Inflammation after Ischemia/Reperfusion.

Mitochondrial inner membrane protein (Mitofilin/Mic60) is part of a big complex that constituent the mitochondrial inner membrane organizing system (MINOS), which plays a critical role in maintaining mitochondrial architecture and function. We recently showed that Mitofilin physically binds to Cyclophilin D, and disruption of this interaction promotes the opening of mitochondrial permeability transition pore (mPTP) and determines the extent of I/R injury. Here, we investigated whether Mitofilin knockout in the mouse enhances myocardial injury and inflammation after I/R injury. We found that full-body deletion (homozygote) of Mitofilin induces a lethal effect in the offspring and that a single allele expression of Mitofilin is sufficient to rescue the mouse phenotype in normal conditions. Using non-ischemic hearts from wild-type (WT) and Mitofilin+/- (HET) mice, we report that the mitochondria structure and calcium retention capacity (CRC) required to induce the opening of mPTP were similar in both groups. However, the levels of mitochondrial dynamics proteins involved in both fusion/fission, including MFN2, DRP1, and OPA1, were slightly reduced in Mitofilin+/- mice compared to WT. After I/R, the CRC and cardiac functional recovery were reduced while the mitochondria structure was more damaged, and myocardial infarct size was increased in Mitofilin+/- mice compared to WT. Mitofilin+/- mice exhibited an increase in the mtDNA release in the cytosol and ROS production, as well as dysregulated SLC25As (3, 5, 11, and 22) solute carrier function, compared to WT. In addition, Mitofilin+/- mice displayed an increase in the transcript of pro-inflammatory markers, including IL-6, ICAM, and TNF-α. These results suggest that Mitofilin knockdown induces mitochondrial cristae damage that promotes dysregulation of SLC25As solute carriers, leading to an increase in ROS production and reduction in CRC after I/R. These effects are associated with an increase in the mtDNA release into the cytosol, where it activates signaling cascades leading to nuclear transcription of pro-inflammatory cytokines that aggravate I/R injury.

RIP3 Translocation into Mitochondria Promotes Mitofilin Degradation to Increase Inflammation and Kidney Injury after Renal Ischemia-Reperfusion.

The receptor-interacting protein kinase 3 (RIP3) has been reported to regulate programmed necrosis-necroptosis forms of cell death with important functions in inflammation. We investigated whether RIP3 translocates into mitochondria in response to renal ischemia-reperfusion (I/R) to interact with inner mitochondrial protein (Mitofilin) and promote mtDNA release into the cytosol. We found that release of mtDNA activates the cGAS-STING pathway, leading to increased nuclear transcription of pro-inflammatory markers that exacerbate renal I/R injury. Monolateral C57/6N and RIP3-/- mice kidneys were subjected to 60 min of ischemia followed by either 12, 24, or 48 h of reperfusion. In WT mice, we found that renal I/R injury increased RIP3 levels, as well as its translocation into mitochondria. We observed that RIP3 interacts with Mitofilin, likely promoting its degradation, resulting in increased mitochondria damage and mtDNA release, activation of the cGAS-STING-p65 pathway, and increased transcription of pro-inflammatory markers. All of these effects observed in WT mice were decreased in RIP3-/- mice. In HK-2, RIP3 overexpression or Mitofilin knockdown increased cell death by activating the cGAS-STING-p65 pathway. Together, this study point to an important role of the RIP3-Mitofilin axis in the initiation and development of renal I/R injury.

Drosophila tubulin polymerization promoting protein mutants reveal pathological correlates relevant to human Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder with no known cure. PD is characterized by locomotion deficits, nigrostriatal dopaminergic neuronal loss, mitochondrial dysfunctions and formation of α-Synuclein aggregates. A well-conserved and less understood family of Tubulin Polymerization Promoting Proteins (TPPP) is also implicated in PD and related disorders, where TPPP exists in pathological aggregates in neurons in patient brains. However, there are no in vivo studies on mammalian TPPP to understand the genetics and neuropathology linking TPPP aggregation or neurotoxicity to PD. Recently, we discovered the only Drosophila homolog of human TPPP named Ringmaker (Ringer). Here, we report that adult ringer mutants display progressive locomotor disabilities, reduced lifespan and neurodegeneration. Importantly, our findings reveal that Ringer is associated with mitochondria and ringer mutants have mitochondrial structural damage and dysfunctions. Adult ringer mutants also display progressive loss of dopaminergic neurons. Together, these phenotypes of ringer mutants recapitulate some of the salient features of human PD patients, thus allowing us to utilize ringer mutants as a fly model relevant to PD, and further explore its genetic and molecular underpinnings to gain insights into the role of human TPPP in PD.

Homoarginine ameliorates diabetic nephropathy independent of nitric oxide synthase-3.

Recently we showed that homoarginine supplementation confers kidney protection in diabetic mouse models. In this study we tested whether the protective effect of homoarginine is nitric oxide synthase-3 (NOS3)-independent in diabetic nephropathy (DN). Experiments were conducted in NOS3 deficient (NOS3-/- ) mice and their wild type littermate using multiple low doses of vehicle or streptozotocin and treated with homoarginine via drinking water for 24 weeks. Homoarginine supplementation for 24 weeks in diabetic NOS3-/- mice significantly attenuated albuminuria, increased blood urea nitrogen, histopathological changes and kidney fibrosis, kidney fibrotic markers, and kidney macrophage recruitment compared with vehicle-treated diabetic NOS3-/- mice. Furthermore, homoarginine supplementation restored kidney mitochondrial function following diabetes. Importantly, there were no significant changes in kidney NOS1 or NOS2 mRNA expression between all groups. In addition, homoarginine supplementation improved cardiac function and reduced cardiac fibrosis following diabetes. These data demonstrate that the protective effect of homoarginine is independent of NOS3, which will ultimately change our understanding of the mechanism(s) by which homoarginine induce renal and cardiac protection in DN. Homoarginine protective effect in DN could be mediated via improving mitochondrial function.

Cardiac ischemia/reperfusion stress reduces inner mitochondrial membrane protein (mitofilin) levels during early reperfusion.

Mitochondrial inner membrane protein (Mitofilin or Mic60) is a mitochondria-shaping protein that plays a key role in maintaining mitochondrial cristae structure and remodeling. We recently showed that Mitofilin knockdown in H9c2 myoblasts induces mitochondrial structural damage resulting in mitochondrial dysfunction that is responsible for cell death via apoptosis. Here, we investigated the role of Mitofilin regulation in ischemia/reperfusion (I/R) injury and studied the relationship between Mitofilin and Cyclophilin (CypD), a key regulator of mitochondrial permeability transition pore (mPTP) opening. C57Bl6 male mice hearts were subjected to different ischemia times (15, 30, or 45 min) followed by a 2 h reperfusion period, or 45 min ischemia followed by 0, 15, 30, 60, or 120 min reperfusion to determine the impact of ischemia or reperfusion times on Mitofilin levels and its interaction with CypD. We found that the increase in myocardial infarct size and the reduction of mitochondrial calcium retention capacity were concomitant with Mitofilin reduction as a function of ischemic duration. We also found that 15 min reperfusion after 45 min ischemia was sufficient to cause a reduction of Mitofilin levels compared to sham, while 45 min ischemia alone was not enough to cause a significant decrease of Mitofilin. We revealed that the c-terminus coiled-coiled domain of Mitofilin is important for its interaction with CypD and the deletion of this identified sequence resulted in a loss of Mitofilin-CypD link, dissipation of mitochondrial membrane potential and increase in cell death. A decrease of the levels of Mitofilin was also associated with mitochondrial structural integrity damage, increased reactive oxygen species (ROS) production, and calpain activity. Our results indicate that Mitofilin physically binds to CypD in the inner mitochondrial membrane and the disruption of this interaction may play a critical role in the increase of mitochondrial dysfunction and initiation of myocytes' death after I/R injury.

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).

Parkin interacts with Mitofilin to increase dopaminergic neuron death in response to Parkinson's disease-related stressors.

Mitochondrial dysfunction plays a critical role in the pathophysiology of Parkinson's disease (PD). The inner mitochondrial membrane (IMM) protein, Mitofilin or Mic60, has been shown to play a key role in controlling and maintaining mitochondrial cristae morphology, and its dysregulation induces cyto-deleterious effects. Here, we investigated the mechanism underlying Mitofilin degradation in dopaminergic neuron death using N27-A cells, and Human Dopamine Neuronal Primary cells treated with PD stressors, Dopamine (DA) or Rotenone (Rot). We found that both PD stressors increased mitochondrial Parkin translocation and interaction with Mitofilin that promotes Mitofilin degradation via ubiquitination, which is responsible for reduced mitochondrial membrane potential and increased ROS production. These effects were concomitant with abnormal mitochondrial structure and increased neuronal death. DA-induced degradation of Mitofilin enhances mitochondrial calpain activity, increases the release of AIF into the cytosol, and promotes apoptosis via an AIF-PARP dependent mechanism. We found that Rot-treated cells exhibit excessive mitophagy, while DA does not trigger mitophagy. In addition, overexpressing USP30, a mitochondrial deubiquitinase, attenuated cell death induced by Rot, but not by DA-treated cells. Together, our study reveals the impact of Parkin-Mitofilin interaction in PD stressor-induced neurotoxicity, which leads to the degradation of Mitofilin, resulting in mitochondrial structural damage and dysfunction that is responsible for neuronal death by apoptosis via an AIF-PARP pathway.

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.

Chloride channel blocker IAA-94 increases myocardial infarction by reducing calcium retention capacity of the cardiac mitochondria.

Indanyloxyacetic acid-94 (IAA-94), an intracellular chloride channel blocker, is shown to ablate cardioprotection rendered by ischemic preconditioning (IPC), N (6)-2-(4-aminophenyl) ethyladenosine or the PKC activator phorbol 12-myristate 13-acetate and cyclosporin A (CsA) in both ex-vivo and in-vivo ischemia-reperfusion (IR) injury. Thus signifying the role of the IAA-94 sensitive chloride channels in mediating cardio-protection upon IR injury. Although IAA-94 sensitive chloride currents are recorded in cardiac mitoplast, there is still a lack of understanding of the mechanism by which IAA-94 increases myocardial infarction (MI) by IR injury. Mitochondria are the key arbitrators of cell life and death pathways. Both oxidative stress and calcium overload in the mitochondria, elicit pathways resulting in the opening of mitochondrial permeability transition pore (mPTP) leading to cell death. Therefore, in this study we explored the role of IAA-94 in MI and in maintaining calcium retention capacity (CRC) of cardiac mitochondria after IR. IAA-94 inhibited the CRC of the isolated cardiac mitochondria in a concentration-dependent manner as measured spectrofluorimetrically using calcium green-5 N. Interestingly, IAA-94 did not change the mitochondrial membrane potential. Further, CsA a blocker of mPTP opening could not override the effect of IAA-94. We also showed for the first time that IAA-94 perfusion after ischemic event augments MI by reducing the CRC of mitochondria. To conclude, our results demonstrate that the mechanism of IAA-94 mediated cardio-deleterious effects is via modulating the mitochondria CRC, thereby playing a role in mPTP opening. These findings highlight new pharmacological targets, which can mediate cardioprotection from IR injury.

Estrogen rescues heart failure through estrogen receptor Beta activation.

BACKGROUND: Recently, we showed that exogenous treatment with estrogen (E2) rescues pre-existing advanced heart failure (HF) in mice. Since most of the biological actions of E2 are mediated through the classical estrogen receptors alpha (ERα) and/or beta (ERß), and both these receptors are present in the heart, we examined the role of ERα and ERß in the rescue action of E2 against HF. METHODS: Severe HF was induced in male mice by transverse aortic constriction-induced pressure overload. Once the ejection fraction (EF) reached ~ 35%, mice were treated with selective agonists for ERα (PPT, 850 µg/kg/day), ERß (DPN, 850 µg/kg/day), or E2 (30 µg/kg/day) together with an ERß-antagonist (PHTPP, 850 µg/kg/day) for 10 days. RESULTS: EF of HF mice was significantly improved to 45.3 ± 2.1% with diarylpropionitrile (DPN) treatment, but not with PPT (31.1 ± 2.3%). E2 failed to rescue HF in the presence of PHTPP, as there was no significant improvement in the EF at the end of the 10-day treatment (32.5 ± 5.2%). The improvement of heart function in HF mice treated with ERß agonist DPN was also associated with reduced cardiac fibrosis and increased cardiac angiogenesis, while the ERα agonist PPT had no significant effect on either cardiac fibrosis or angiogenesis. Furthermore, DPN improved hemodynamic parameters in HF mice, whereas PPT had no significant effect. CONCLUSIONS: E2 treatment rescues pre-existing severe HF mainly through ERß. Rescue of HF by ERß activation is also associated with stimulation of cardiac angiogenesis, suppression of fibrosis, and restoration of hemodynamic parameters.

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.

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.

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.

Rescue of Pressure Overload-Induced Heart Failure by Estrogen Therapy.

BACKGROUND: Estrogen pretreatment has been shown to attenuate the development of heart hypertrophy, but it is not known whether estrogen could also rescue heart failure (HF). Furthermore, the heart has all the machinery to locally biosynthesize estrogen via aromatase, but the role of local cardiac estrogen synthesis in HF has not yet been studied. Here we hypothesized that cardiac estrogen is reduced in HF and examined whether exogenous estrogen therapy can rescue HF. METHODS AND RESULTS: HF was induced by transaortic constriction in mice, and once mice reached an ejection fraction (EF) of ≈35%, they were treated with estrogen for 10 days. Cardiac structure and function, angiogenesis, and fibrosis were assessed, and estrogen was measured in plasma and in heart. Cardiac estrogen concentrations (6.18±1.12 pg/160 mg heart in HF versus 17.79±1.28 pg/mL in control) and aromatase transcripts (0.19±0.04, normalized to control, P<0.05) were significantly reduced in HF. Estrogen therapy increased cardiac estrogen 3-fold and restored aromatase transcripts. Estrogen also rescued HF by restoring ejection fraction to 53.1±1.3% (P<0.001) and improving cardiac hemodynamics both in male and female mice. Estrogen therapy stimulated angiogenesis as capillary density increased from 0.66±0.07 in HF to 2.83±0.14 (P<0.001, normalized to control) and reversed the fibrotic scarring observed in HF (45.5±2.8% in HF versus 5.3±1.0%, P<0.001). Stimulation of angiogenesis by estrogen seems to be one of the key mechanisms, since in the presence of an angiogenesis inhibitor estrogen failed to rescue HF (ejection fraction=29.3±2.1%, P<0.001 versus E2). CONCLUSIONS: Estrogen rescues pre-existing HF by restoring cardiac estrogen and aromatase, stimulating angiogenesis, and suppressing fibrosis.