High-mobility group box 1 increases platelet surface P2Y12 and platelet activation in sickle cell disease.

Thrombosis and inflammation are intimately linked and synergistically contribute to the pathogenesis of numerous thromboinflammatory diseases, including sickle cell disease (SCD). While platelets are central to thrombogenesis and inflammation, the molecular mechanisms of crosstalk between the 2 remain elusive. High-mobility group box 1 (HMGB1) regulates inflammation and stimulates platelet activation through Toll-like receptor 4. However, it remains unclear whether HMGB1 modulates other thrombotic agonists to regulate platelet activation. Herein, using human platelets, we demonstrate that HMGB1 significantly enhanced ADP-mediated platelet activation. Furthermore, inhibition of the purinergic receptor P2Y12 attenuated HMGB1-dependent platelet activation. Mechanistically, we show that HMGB1 stimulated ADP secretion, while concomitantly increasing P2Y12 levels at the platelet membrane. We show that in SCD patients, increased plasma HMGB1 levels were associated with heightened platelet activation and surface P2Y12 expression. Treatment of healthy platelets with plasma from SCD patients enhanced platelet activation and surface P2Y12, and increased sensitivity to ADP-mediated activation, and these effects were linked to plasma HMGB1. We conclude that HMGB1-mediated platelet activation involves ADP-dependent P2Y12 signaling, and HMGB1 primes platelets for ADP signaling. This complementary agonism between ADP and HMGB1 furthers the understanding of thromboinflammatory signaling in conditions such as SCD, and provides insight for therapeutic P2Y12 inhibition.

Acoustic Activation Imaging With Intravenous Perfluoropropane Nanodroplets Results in Selective Bioactivation of the Risk Area.

BACKGROUND: Acoustically activatable perfluoropropane droplets (PD) can be formulated from commercially available microbubble preparations. Diagnostic transthoracic ultrasound frequencies have resulted in acoustic activation (AA) predominately within myocardial infarct zones (IZ). OBJECTIVE: We hypothesized that the AA area following acute coronary ischemia/reperfusion (I/R) would selectively enhance the developing scar zone, and target bioeffects specifically to this region. METHODS: We administered intravenous PD in 36 rats and 20 pigs at various stages of myocardial scar formation (30 minutes, 1 day, and 7 days post I/R) to determine what effect infarct age had on the AA within the IZ. This was correlated with histology, myeloperoxidase activity, and tissue nitrite activity. RESULTS: The degree of AA within the IZ in rats was not associated with collagen content, neutrophil infiltration, or infarct age. AA within 24 hours of I/R was associated with increased nitric oxide utilization selectively within the IZ (P < .05 compared with remote zone). The spatial extent of AA in pigs correlated with infarct size only when performed before sacrifice at 7 days (r = .74, P < .01). CONCLUSIONS: Acoustic activation of intravenous PD enhances the developing scar zone following I/R, and results in selective tissue nitric oxide utilization.

Myoglobin modulates the Hippo pathway to promote cardiomyocyte differentiation.

The endogenous mechanisms that propagate cardiomyocyte differentiation and prevent de-differentiation remain unclear. While the expression of the heme protein myoglobin increases by over 50% during cardiomyocyte differentiation, a role for myoglobin in regulating cardiomyocyte differentiation has not been tested. Here, we show that deletion of myoglobin in cardiomyocyte models decreases the gene expression of differentiation markers and stimulates cellular proliferation, consistent with cardiomyocyte de-differentiation. Mechanistically, the heme prosthetic group of myoglobin catalyzes the oxidation of the Hippo pathway kinase LATS1, resulting in phosphorylation and inactivation of yes-associated protein (YAP). In vivo, myoglobin-deficient zebrafish hearts show YAP dephosphorylation and accelerated cardiac regeneration after apical injury. Similarly, myoglobin knockdown in neonatal murine hearts shows increased YAP dephosphorylation and cardiomyocyte cycling. These data demonstrate a novel role for myoglobin as an endogenous driver of cardiomyocyte differentiation and highlight myoglobin as a potential target to enhance cardiac development and improve cardiac repair and regeneration.

Allele-specific control of rodent and human lncRNA KMT2E-AS1 promotes hypoxic endothelial pathology in pulmonary hypertension.

Hypoxic reprogramming of vasculature relies on genetic, epigenetic, and metabolic circuitry, but the control points are unknown. In pulmonary arterial hypertension (PAH), a disease driven by hypoxia inducible factor (HIF)-dependent vascular dysfunction, HIF-2α promoted expression of neighboring genes, long noncoding RNA (lncRNA) histone lysine N-methyltransferase 2E-antisense 1 (KMT2E-AS1) and histone lysine N-methyltransferase 2E (KMT2E). KMT2E-AS1 stabilized KMT2E protein to increase epigenetic histone 3 lysine 4 trimethylation (H3K4me3), driving HIF-2α-dependent metabolic and pathogenic endothelial activity. This lncRNA axis also increased HIF-2α expression across epigenetic, transcriptional, and posttranscriptional contexts, thus promoting a positive feedback loop to further augment HIF-2α activity. We identified a genetic association between rs73184087, a single-nucleotide variant (SNV) within a KMT2E intron, and disease risk in PAH discovery and replication patient cohorts and in a global meta-analysis. This SNV displayed allele (G)-specific association with HIF-2α, engaged in long-range chromatin interactions, and induced the lncRNA-KMT2E tandem in hypoxic (G/G) cells. In vivo, KMT2E-AS1 deficiency protected against PAH in mice, as did pharmacologic inhibition of histone methylation in rats. Conversely, forced lncRNA expression promoted more severe PH. Thus, the KMT2E-AS1/KMT2E pair orchestrates across convergent multi-ome landscapes to mediate HIF-2α pathobiology and represents a key clinical target in pulmonary hypertension.

Mitochondria-containing extracellular vesicles from mouse vs . human brain endothelial cells for ischemic stroke therapy.

Ischemic stroke-induced mitochondrial dysfunction in the blood-brain barrier-forming brain endothelial cells ( BECs ) results in long-term neurological dysfunction post-stroke. We previously reported that intravenous administration of human BEC ( hBEC )-derived mitochondria-containing extracellular vesicles ( EVs ) showed a potential efficacy signal in a mouse middle cerebral artery occlusion ( MCAo ) model of stroke. We hypothesized that EVs harvested from donor species homologous to the recipient species ( e.g., mouse) may improve therapeutic efficacy, and therefore, use of mouse BEC ( mBEC )-derived EVs may improve post-stroke outcomes in MCAo mice. We investigated if EVs derived from the same species as the recipient cell (mBEC-EVs and recipient mBECs or hBECs-EVs and recipient hBECs) show a greater EV mitochondria delivery efficiency than cross-species EVs and recipient cells (mBEC-EVs and recipient hBECs or vice versa ). Our results showed that mBEC-EVs outperformed hBEC-EVs in transferring EV mitochondria to the recipient ischemic mBECs, and improved mBEC mitochondrial function via increasing oxygen consumption rate. mBEC-EVs significantly reduced brain infarct volume and improved behavioral recovery compared to vehicle-injected MCAo mice. Our data suggests that mBEC-EVs show superior therapeutic efficacy in a mouse MCAo stroke model compared to hBEC-EVs-supporting the continued use of mBEC-EVs to optimize the therapeutic potential of mitochondria-containing EVs in preclinical studies.

An ERK5-PFKFB3 axis regulates glycolysis and represents a therapeutic vulnerability in pediatric diffuse midline glioma.

Metabolic reprogramming in pediatric diffuse midline glioma is driven by gene expression changes induced by the hallmark histone mutation H3K27M, which results in aberrantly permissive activation of oncogenic signaling pathways. Previous studies of diffuse midline glioma with altered H3K27 (DMG-H3K27a) have shown that the RAS pathway, specifically through its downstream kinase, extracellular-signal-related kinase 5 (ERK5), is critical for tumor growth. Further downstream effectors of ERK5 and their role in DMG-H3K27a metabolic reprogramming have not been explored. We establish that ERK5 is a critical regulator of cell proliferation and glycolysis in DMG-H3K27a. We demonstrate that ERK5 mediates glycolysis through activation of transcription factor MEF2A, which subsequently modulates expression of glycolytic enzyme PFKFB3. We show that in vitro and mouse models of DMG-H3K27a are sensitive to the loss of PFKFB3. Multi-targeted drug therapy against the ERK5-PFKFB3 axis, such as with small-molecule inhibitors, may represent a promising therapeutic approach in patients with pediatric diffuse midline glioma.

High Dose of Metformin Decreases Susceptibility to Occlusive Arterial Thrombosis in Diabetic Mice.

Introduction: Metformin is the most prescribed medication in Diabetes Mellitus(DM). Metformin has shown to decrease mean platelet volume, with promising antiplatelet effects. High doses of Metformin have also been associated with hypercoagulation. We hypothesize that Metformin will protect DM mice from occlusive arterial thrombus formation by altering platelet activation and mitochondrial bioenergetics. Methods: DM was developed by low dose of Streptozotocin, non-DM (healthy) mice are controls. Either vehicle or Metformin was administered twice daily via oral gavage for 7-days. Ferric chloride (FeCl3) arterial thrombosis and tail bleeding time were performed. Whole blood aggregometry, platelet activation/adhesion and mitochondrial bioenergetics were evaluated. Results: Metformin decreased susceptibility of DM mice to arterial thrombosis. Platelet bioenergetics show DM mice have increased platelet mitochondrial respiration, but no differences were observed with Metformin treatment. In non-DM (healthy) mice, Metformin modulated ADP-dependent increase in platelet adhesion. Non-DM (healthy) mice, Metformin shortens bleeding time with faster thrombotic occlusion. Metformin also increased platelet mitochondrial maximal respiration and spare respiratory capacity uniquely in non-DM (healthy) mice. Conclusion: Metformin regulates platelet bioenergetics and ADP-mediated platelet function in DM mice which attenuates susceptibility to arterial thrombosis. Future studies will evaluate clinically relevant doses of Metformin that regulates thrombotic function in diabetic platelets.

A Comprehensive Exercise (COMEX) Intervention to Optimize Exercise Participation for Improving Patient-Centered Outcomes and Physical Functioning in Patients Receiving Hemodialysis: Development and Pilot Testing.

Rationale & Objective: To address the need for an intradialytic exercise program that is easily delivered in clinical setting, engaging and scalable, we developed a novel COMprehensive EXercise (COMEX) program based on input from patients receiving hemodialysis (HD), dialysis staff members and nephrologists. The objective of this study was to determine the feasibility, safety, and acceptance of COMEX during HD. Study Design: Single-arm prospective pilot feasibility study. Setting & Participants: Seventeen patients receiving in-center HD. Intervention: Three-month participation in the COMEX program, which included video-based dialysis chair exercises (aerobic and resistance) integrated with educational and motivational components. Outcomes: Data on recruitment, adherence, safety and acceptability were collected. Additional assessments were performed to evaluate changes in physical functioning, patient-reported symptoms, and objectively measured sleep and physical activity. We also examined the feasibility of obtaining skeletal muscle biopsies and blood samples to explore molecular mechanisms of muscle atrophy and to assess platelet mitochondrial function and adaptation to exercise during HD. Results: Thirteen of the 17 (76%) participants completed the 3-month intervention. The mean participant age was 63.6 ± 15.1 years. In total, 46% of participants were males, and 55% were White. The mean body mass index was 38.7 ± 11.6 kg/m2. There were no reported adverse effects, and the adherence rate to exercise sessions was high with 88% of the sessions completed. Patient satisfaction was high, as 100% of the patients would recommend the program to other dialysis patients. It was feasible to collect data on physical functioning, patient-reported symptoms, and objective sleep and physical activity and to obtain muscle biopsies and blood samples. Limitations: Small sample size, lack of an onsite exercise professional, and technological issues with telemedicine behavioral motivation. Conclusions: The COMEX intradialytic exercise intervention is safe and acceptable to patients, and outcome measures were feasible to obtain. Future studies should consider including exercise professionals to facilitate progression through a personalized exercise protocol. Funding Source: This work is supported by pilot award from P30 DK079307 (PI, Jhamb). Trial Registration: ClinicalTrials.gov, NCT03055299. Plain-Language Summary: We tested a new COMprehensive EXercise (COMEX) program to deliver exercise during dialysis. This 3-month program included video-based dialysis chair exercises (aerobic and resistance) integrated with educational and motivational components. Our study shows COMEX was feasible, had high satisfaction and adherence, and was safe. It was feasible to collect data on physical functioning, patient-reported symptoms, and objective sleep and physical activity and to obtain muscle biopsies and blood samples. Future studies should consider including exercise professionals to facilitate progression through a personalized exercise protocol.

Interferon signaling drives epithelial metabolic reprogramming to promote secondary bacterial infection.

Clinical studies report that viral infections promote acute or chronic bacterial infections at multiple host sites. These viral-bacterial co-infections are widely linked to more severe clinical outcomes. In experimental models in vitro and in vivo, virus-induced interferon responses can augment host susceptibility to secondary bacterial infection. Here, we used a cell-based screen to assess 389 interferon-stimulated genes (ISGs) for their ability to induce chronic Pseudomonas aeruginosa infection. We identified and validated five ISGs that were sufficient to promote bacterial infection. Furthermore, we dissected the mechanism of action of hexokinase 2 (HK2), a gene involved in the induction of aerobic glycolysis, commonly known as the Warburg effect. We report that HK2 upregulation mediates the induction of Warburg effect and secretion of L-lactate, which enhances chronic P. aeruginosa infection. These findings elucidate how the antiviral immune response renders the host susceptible to secondary bacterial infection, revealing potential strategies for viral-bacterial co-infection treatment.

A blood-based marker of mitochondrial DNA damage in Parkinson's disease.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder, and neuroprotective or disease-modifying interventions remain elusive. High-throughput markers aimed at stratifying patients on the basis of shared etiology are required to ensure the success of disease-modifying therapies in clinical trials. Mitochondrial dysfunction plays a prominent role in the pathogenesis of PD. Previously, we found brain region-specific accumulation of mitochondrial DNA (mtDNA) damage in PD neuronal culture and animal models, as well as in human PD postmortem brain tissue. To investigate mtDNA damage as a potential blood-based marker for PD, we describe herein a PCR-based assay (Mito DNADX) that allows for the accurate real-time quantification of mtDNA damage in a scalable platform. We found that mtDNA damage was increased in peripheral blood mononuclear cells derived from patients with idiopathic PD and those harboring the PD-associated leucine-rich repeat kinase 2 (LRRK2) G2019S mutation in comparison with age-matched controls. In addition, mtDNA damage was elevated in non-disease-manifesting LRRK2 mutation carriers, demonstrating that mtDNA damage can occur irrespective of a PD diagnosis. We further established that Lrrk2 G2019S knock-in mice displayed increased mtDNA damage, whereas Lrrk2 knockout mice showed fewer mtDNA lesions in the ventral midbrain, compared with wild-type control mice. Furthermore, a small-molecule kinase inhibitor of LRRK2 mitigated mtDNA damage in a rotenone PD rat midbrain neuron model and in idiopathic PD patient-derived lymphoblastoid cell lines. Quantifying mtDNA damage using the Mito DNADX assay may have utility as a candidate marker of PD and for measuring the pharmacodynamic response to LRRK2 kinase inhibitors.

Lysosomal mitochondrial interaction promotes tumor growth in squamous cell carcinoma of the head and neck.

Tumor growth and proliferation are regulated by numerous mechanisms. Communication between intracellular organelles has recently been shown to regulate cellular proliferation and fitness. The way lysosomes and mitochondria communicate with each other (lysosomal/mitochondrial interaction) is emerging as a major determinant of tumor proliferation and growth. About 30% of squamous carcinomas (including squamous cell carcinoma of the head and neck, SCCHN) overexpress TMEM16A, a calcium-activated chloride channel, which promotes cellular growth and negatively correlates with patient survival. TMEM16A has recently been shown to drive lysosomal biogenesis, but its impact on mitochondrial function is unclear. Here, we show that (1) patients with high TMEM16A SCCHN display increased mitochondrial content specifically complex I; (2) In vitro and in vivo models uniquely depend on mitochondrial complex I activity for growth and survival; (3) ß-catenin/NRF2 signaling is a critical linchpin that drives mitochondrial biogenesis, and (4) mitochondrial complex I and lysosomal function are codependent for proliferation. Taken together, our data demonstrate that LMI drives tumor proliferation and facilitates a functional interaction between lysosomes and mitochondria. Therefore, inhibition of LMI may serve as a therapeutic strategy for patients with SCCHN.

High Dose of Metformin Decreases Susceptibility to Occlusive Arterial Thrombosis in Diabetic Mice.

Introduction: Metformin is the most prescribed medication in Diabetes Mellitus(DM). Metformin has shown to decrease mean platelet volume, with promising antiplatelet effects. High doses of Metformin have also been associated with hypercoagulation. We hypothesize that Metformin will protect DM mice from occlusive arterial thrombus formation by altering platelet activation and mitochondrial bioenergetics. Methods: DM was developed by low dose of Streptozotocin, healthy (non-DM) mice are controls. Either vehicle or Metformin was administered twice daily via oral gavage for 7-days. Ferric chloride (FeCl3) arterial thrombosis and tail bleeding time were performed. Whole blood aggregometry, platelet activation/adhesion and mitochondrial bioenergetics were evaluated. Results: Metformin decreased susceptibility of DM mice to arterial thrombosis. Platelet bioenergetics show DM mice have increased platelet mitochondrial respiration, but no differences were observed with Metformin treatment. In healthy mice, Metformin modulated ADP-dependent increase in platelet adhesion. In healthy mice, Metformin shortens bleeding time with faster thrombotic occlusion. Metformin also increased platelet mitochondrial maximal respiration and spare respiratory capacity uniquely in healthy mice. Conclusion: Metformin regulates platelet bioenergetics and ADP-mediated platelet function in DM mice which attenuates susceptibility to arterial thrombosis. Future studies will evaluate clinically relevant doses of Metformin that regulates thrombotic function in diabetic platelets.

Reduced acetylation of TFAM promotes bioenergetic dysfunction in the failing heart.

General control of amino acid synthesis 5-like 1 (GCN5L1) was previously identified as a key regulator of protein lysine acetylation in mitochondria. Subsequent studies demonstrated that GCN5L1 regulates the acetylation status and activity of mitochondrial fuel substrate metabolism enzymes. However, the role of GCN5L1 in response to chronic hemodynamic stress is largely unknown. Here, we show that cardiomyocyte-specific GCN5L1 knockout mice (cGCN5L1 KO) display exacerbated heart failure progression following transaortic constriction (TAC). Mitochondrial DNA and protein levels were decreased in cGCN5L1 KO hearts after TAC, and isolated neonatal cardiomyocytes with reduced GCN5L1 expression had lower bioenergetic output in response to hypertrophic stress. Loss of GCN5L1 expression led to a decrease in the acetylation status of mitochondrial transcription factor A (TFAM) after TAC in vivo, which was linked to a reduction in mtDNA levels in vitro. Together, these data suggest that GCN5L1 may protect from hemodynamic stress by maintaining mitochondrial bioenergetic output.

Frataxin deficiency disrupts mitochondrial respiration and pulmonary endothelial cell function.

Deficiency of iron­sulfur (FeS) clusters promotes metabolic rewiring of the endothelium and the development of pulmonary hypertension (PH) in vivo. Joining a growing number of FeS biogenesis proteins critical to pulmonary endothelial function, recent data highlighted that frataxin (FXN) reduction drives Fe-S-dependent genotoxic stress and senescence across multiple types of pulmonary vascular disease. Trinucleotide repeat mutations in the FXN gene cause Friedreich's ataxia, a disease characterized by cardiomyopathy and neurodegeneration. These tissue-specific phenotypes have historically been attributed to mitochondrial reprogramming and oxidative stress. Whether FXN coordinates both nuclear and mitochondrial processes in the endothelium is unknown. Here, we aim to identify the mitochondria-specific effects of FXN deficiency in the endothelium that predispose to pulmonary hypertension. Our data highlight an Fe-S-driven metabolic shift separate from previously described replication stress whereby FXN knockdown diminished mitochondrial respiration and increased glycolysis and oxidative species production. In turn, FXN-deficient endothelial cells had increased vasoconstrictor production (EDN1) and decreased nitric oxide synthase expression (NOS3). These data were observed in primary pulmonary endothelial cells after pharmacologic inhibition of FXN, mice carrying a genetic endothelial deletion of FXN, and inducible pluripotent stem cell-derived endothelial cells from patients with FXN mutations. Altogether, this study indicates FXN is an upstream driver of pathologic aberrations in metabolism and genomic stability. Moreover, our study highlights FXN-specific vasoconstriction in vivo, prompting future studies to investigate available and novel PH therapies in contexts of FXN deficiency.

Release of hepatic xanthine oxidase (XO) to the circulation is protective in intravascular hemolytic crisis.

Xanthine oxidase (XO) catalyzes the catabolism of hypoxanthine to xanthine and xanthine to uric acid, generating oxidants as a byproduct. Importantly, XO activity is elevated in numerous hemolytic conditions including sickle cell disease (SCD); however, the role of XO in this context has not been elucidated. Whereas long-standing dogma suggests elevated levels of XO in the vascular compartment contribute to vascular pathology via increased oxidant production, herein, we demonstrate, for the first time, that XO has an unexpected protective role during hemolysis. Using an established hemolysis model, we found that intravascular hemin challenge (40 µmol/kg) resulted in a significant increase in hemolysis and an immense (20-fold) elevation in plasma XO activity in Townes sickle cell phenotype (SS) sickle mice compared to controls. Repeating the hemin challenge model in hepatocyte-specific XO knockout mice transplanted with SS bone marrow confirmed the liver as the source of enhanced circulating XO as these mice demonstrated 100% lethality compared to 40% survival in controls. In addition, studies in murine hepatocytes (AML12) revealed hemin mediates upregulation and release of XO to the medium in a toll like receptor 4 (TLR4)-dependent manner. Furthermore, we demonstrate that XO degrades oxyhemoglobin and releases free hemin and iron in a hydrogen peroxide-dependent manner. Additional biochemical studies revealed purified XO binds free hemin to diminish the potential for deleterious hemin-related redox reactions as well as prevents platelet aggregation. In the aggregate, data herein reveals that intravascular hemin challenge induces XO release by hepatocytes through hemin-TLR4 signaling, resulting in an immense elevation of circulating XO. This increased XO activity in the vascular compartment mediates protection from intravascular hemin crisis by binding and potentially degrading hemin at the apical surface of the endothelium where XO is known to be bound and sequestered by endothelial glycosaminoglycans (GAGs).

Mitochondria-containing extracellular vesicles (EV) reduce mouse brain infarct sizes and EV/HSP27 protect ischemic brain endothelial cultures.

Ischemic stroke causes brain endothelial cell (BEC) death and damages tight junction integrity of the blood-brain barrier (BBB). We harnessed the innate mitochondrial load of BEC-derived extracellular vesicles (EVs) and utilized mixtures of EV/exogenous 27 kDa heat shock protein (HSP27) as a one-two punch strategy to increase BEC survival (via EV mitochondria) and preserve their tight junction integrity (via HSP27 effects). We demonstrated that the medium-to-large (m/lEV) but not small EVs (sEV) transferred their mitochondrial load, that subsequently colocalized with the mitochondrial network of the recipient primary human BECs. Recipient BECs treated with m/lEVs showed increased relative ATP levels and mitochondrial function. To determine if the m/lEV-meditated increase in recipient BEC ATP levels was associated with m/lEV mitochondria, we isolated m/lEVs from donor BECs pre-treated with oligomycin A (OGM, mitochondria electron transport complex V inhibitor), referred to as OGM-m/lEVs. BECs treated with naïve m/lEVs showed a significant increase in ATP levels compared to untreated OGD cells, OGM-m/lEVs treated BECs showed a loss of ATP levels suggesting that the m/lEV-mediated increase in ATP levels is likely a function of their innate mitochondrial load. In contrast, sEV-mediated ATP increases were not affected by inhibition of mitochondrial function in the donor BECs. Intravenously administered m/lEVs showed a reduction in brain infarct sizes compared to vehicle-injected mice in a mouse middle cerebral artery occlusion model of ischemic stroke. We formulated binary mixtures of human recombinant HSP27 protein with EVs: EV/HSP27 and ternary mixtures of HSP27 and EVs with a cationic polymer, poly (ethylene glycol)-b-poly (diethyltriamine): (PEG-DET/HSP27)/EV. (PEG-DET/HSP27)/EV and EV/HSP27 mixtures decreased the paracellular permeability of small and large molecular mass fluorescent tracers in oxygen glucose-deprived primary human BECs. This one-two punch approach to increase BEC metabolic function and tight junction integrity may be a promising strategy for BBB protection and prevention of long-term neurological dysfunction post-ischemic stroke.

Myocardial brain-derived neurotrophic factor regulates cardiac bioenergetics through the transcription factor Yin Yang 1.

AIMS: Brain-derived neurotrophic factor (BDNF) is markedly decreased in heart failure patients. Both BDNF and its receptor, tropomyosin-related kinase receptor (TrkB), are expressed in cardiomyocytes; however, the role of myocardial BDNF signalling in cardiac pathophysiology is poorly understood. Here, we investigated the role of BDNF/TrkB signalling in cardiac stress response to exercise and pathological stress. METHODS AND RESULTS: We found that myocardial BDNF expression was increased in mice with swimming exercise but decreased in a mouse heart failure model and human failing hearts. Cardiac-specific TrkB knockout (cTrkB KO) mice displayed a blunted adaptive cardiac response to exercise, with attenuated upregulation of transcription factor networks controlling mitochondrial biogenesis/metabolism, including peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α). In response to pathological stress (transaortic constriction, TAC), cTrkB KO mice showed an exacerbated heart failure progression. The downregulation of PGC-1α in cTrkB KO mice exposed to exercise or TAC resulted in decreased cardiac energetics. We further unravelled that BDNF induces PGC-1α upregulation and bioenergetics through a novel signalling pathway, the pleiotropic transcription factor Yin Yang 1. CONCLUSION: Taken together, our findings suggest that myocardial BDNF plays a critical role in regulating cellular energetics in the cardiac stress response.

Aging-Associated Changes in Oxidative Stress Negatively Impacts the Urinary Bladder Urothelium.

PURPOSE: Lower urinary tract symptoms are known to significantly increase with age, negatively impacting quality of life and self-reliance. The urothelium fulfills crucial tasks, serving as a barrier protecting the underlying bladder tissue from the harsh chemical composition of urine, and exhibits signaling properties via the release of mediators within the bladder wall that affect bladder functioning. Aging is associated with detrimental changes in cellular health, in part by increasing oxidative stress in the bladder mucosa, and more specifically the urothelium. This, in turn, may impact urothelial mitochondrial health and bioenergetics. METHODS: We collected mucosal tissue samples from both young (3-4 months old) and aged (25-30 months old) rats. Tissue was evaluated for p21-Arc, nitrotyrosine, and cytochrome C expression by western immunoblotting. Urothelial cells were cultured for single-cell imaging to analyze basal levels of reactive oxygen species and the mitochondrial membrane potential. Mitochondrial bioenergetics and cellular respiration were investigated by the Seahorse assay, and measurements of adenosine triphosphate release were made using the luciferin-luciferase assay. RESULTS: Aging was associated with a significant increase in biomarkers of cellular senescence, oxidative stress, and basal levels of reactive oxygen species. The mitochondrial membrane potential was significantly lower in urothelial cell cultures from aged animals, and cultures from aged animals showed a significant decrease in mitochondrial bioenergetics. CONCLUSION: Aging-related increases in oxidative stress and excessive reactive oxygen species may be contributing factors underlying lower urinary tract symptoms in older adults. The mechanisms outlined in this study could be utilized to identify novel pharmaceutical targets to improve aging-associated bladder dysfunction.