Mitochondrial Profiles Research Articles

An integrated multi-omics approach revealed the regulation of melatonin on age-dependent mitochondrial function impair and lipid dyshomeostasis in mice hippocampus

Melatonin can improve mitochondrial dysfunction associated with the aging process by removing active oxygen, as well as inhibiting lipid peroxidation to maintain biofilm fluidity and resist free radical attack. However, there is poor understanding of the effect of melatonin on age-dependent mitochondrial function and lipid profile changes in brain. In this study, we investigated the energy metabolism of the whole body and brain of mice at 9 months, 13 months, and 25 months of continuous gastric administration of 3 mg/kg/d melatonin once per day morning for two months. In addition, we performed transcriptomic, proteomic and lipidomic analysis in the hippocampus of mice at different ages. Proteomics showed that melatonin regulated mitochondrial electron transport and leucine degradation in mouse hippocampus. Lipomics suggested that the long-chain unsaturated glycerol phospholipids in mouse hippocampus increased in an age-dependent manner, while ceramide and glycerol phospholipids decreased significantly in hippocampus of mouse chronically exposed to melatonin. The combined analysis of proteome and liposome demonstrated that Mpst, Ccsap, Hdhd5, Rpl5 and Flna were the key proteins of the network which involved in the regulation of numerous lipids. Furthermore, ultrastructure observation results illustrated that melatonin could improve the damaged mitochondrial and morphologies of 25-month-old mice hippocampus. In conclusion, we describe a mechanism that age-dependent up-regulation of long-chain unsaturated lipids is a driving risk factor for mitochondrial damage and this effect could be reversed by chronic supplement of low-dose melatonin.

Identification of the metabolic remodeling profile in the early-stage of myocardial ischemia and the contributory role of mitochondrion

ABSTRACT Cardiac remodeling is the primary pathological feature of chronic heart failure. Prompt inhibition of remodeling in acute coronary syndrome has been a standard procedure, but the morbidity and mortality are still high. Exploring the characteristics of ischemia in much earlier stages and identifying its biomarkers are essential for introducing novel mechanisms and therapeutic strategies. Metabolic and structural remodeling of mitochondrion is identified to play key roles in ischemic heart disease. The mitochondrial metabolic features in early ischemia have not previously been described. In the present study, we established a mouse heart in early ischemia and explored the mitochondrial metabolic profile using metabolomics analysis. We also discussed the role of mitochondrion in the global cardiac metabolism. Transmission electron microscopy revealed that mitochondrial structural injury was invoked at 8 minutes post-coronary occlusion. In total, 75 metabolites in myocardium and 26 in mitochondria were screened out. About 23% of the differentiated metabolites in mitochondria overlapped with the differentiated metabolites in myocardium; Total 81% of the perturbed metabolic pathway in mitochondria overlapped with the perturbed pathway in myocardium, and these pathways accounted for 50% of the perturbed pathway in myocardium. Purine metabolism was striking and mechanically important. In conclusion, in the early ischemia, myocardium exacerbated metabolic remodeling. Mitochondrion was a contributor to the myocardial metabolic disorder. Purine metabolism may be a potential biomarker for early ischemia diagnosis. Our study introduced a perspective for prompt identification of ischemia.

Effects of <i>Euphorbia thymifolia</i> and <i>Euphorbia hirta</i> leaf extracts on membrane-bound, mitochondrial enzymes and lipid profile of carbon tetrachloride-induced hepatotoxicity in rats

The present investigation was aimed to identify the potentiality of <i>Euphorbia thymifolia</i> Linn. and <i>Euphorbia hirta</i> Linn. leaf extract on the toxin-induced (carbon tetrachloride - CCl<sub>4</sub>) Albino Wistar rats. The animals were grouped into 7 categories including control (basal diet, G1), CCl<sub>4</sub>-induced (1.5 mL/kg, b.w., i.p.) (G2), G1 administrated with 300 mg/kg b.w., extract of <i>E. thymifolia</i> (G3) and <i>E. hirta</i> (G4), G2 administrated with 300 mg/kg b.w., extract of <i>E. thymifolia</i> (G5), <i>E. hirta</i> (G6), and standard drug (silymarin 25 mg/kg b.w.; G7) for 21-days trial period with each group contains 6 rats. The samples were collected and the following parameters including mitochondrial enzymes, different ATPase and lipid profiles were analyzed. The membrane-bound enzymes, the mitochondrial enzymes levels and the lipid profiles were reduced in the toxin-induced rats but the levels of enzymes were restored, significantly increased and lipid profiles are returned to the normal in the treatment of both extracts.

α-Synuclein at the Presynaptic Axon Terminal as a Double-Edged Sword.

α-synuclein (α-syn) is a presynaptic, lipid-binding protein strongly associated with the neuropathology observed in Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and Alzheimer’s Disease (AD). In normal physiology, α-syn plays a pivotal role in facilitating endocytosis and exocytosis. Interestingly, mutations and modifications of precise α-syn domains interfere with α-syn oligomerization and nucleation that negatively affect presynaptic vesicular dynamics, protein expressions, and mitochondrial profiles. Furthermore, the integration of the α-syn oligomers into the presynaptic membrane results in pore formations, ion influx, and excitotoxicity. Targeted therapies against specific domains of α-syn, including the use of small organic molecules, monoclonal antibodies, and synthetic peptides, are being screened and developed. However, the prospect of an effective α-syn targeted therapy is still plagued by low permeability across the blood–brain barrier (BBB), and poor entry into the presynaptic axon terminals. The present review proposes a modification of current strategies, which includes the use of novel encapsulation technology, such as lipid nanoparticles, to bypass the BBB and deliver such agents into the brain.

Liver-specific overexpression of SIRT3 enhances oxidative metabolism, but does not impact metabolic defects induced by high fat feeding in mice

The mitochondrial enzyme SIRT3 is an NAD+-dependent deacetylase important in cell metabolism, and a decline in its protein expression or activity has been linked with insulin resistance in obesity, ageing and type 2 diabetes. While studies in SIRT3 knockout mice have dramatically improved our understanding of the function of SIRT3, the impact of increasing SIRT3 levels remains under-examined. In this study we investigated the effects of liver-specific SIRT3 overexpression in mice on mitochondrial function and metabolic profile in both isolated hepatocytes and in vivo. Primary hepatocytes overexpressing SIRT3 displayed increased oxygen consumption and a reduction in triglyceride accumulation. In mice with hepatic SIRT3 overexpression, increased fasting β-hydroxybutyrate levels were observed, coupled with an increase in oxygen consumption in isolated mitochondria and increased substrate utilization in liver homogenates. However, metabolic profiling of mice exposed to either chow or high-fat diet revealed no effect of hepatic SIRT3 overexpression on glucose tolerance, body composition or tissue triglyceride accumulation. These findings suggest limited whole-body benefit of increasing hepatic SIRT3 during the development of diet-induced insulin resistance.

Assessment of mitochondrial toxicity in newborns and infants with congenital cytomegalovirus infection treated with valganciclovir

BackgroundGanciclovir/valganciclovir is currently indicated during the first 6 months of life in symptomatic children with congenital cytomegalovirus (CMV) infection. However, this treatment may have the potential to induce mitochondrial toxicity...

Hydrogel-Mediated Release of TRPV1 Modulators to Fine Tune Osteoclastogenesis.

Bone defects, including bone loss due to increased osteoclast activity, have become a global health-related issue. Osteoclasts attach to the bone matrix and resorb the same, playing a vital role in bone remodeling. Ca2+ homeostasis plays a pivotal role in the differentiation and maturation of osteoclasts. In this work, we examined the role of TRPV1, a nonselective cation channel, in osteoclast function and differentiation. We demonstrate that endogenous TRPV1 is functional and causes Ca2+ influx upon activation with pharmacological activators [resiniferatoxin (RTX) and capsaicin] at nanomolar concentration, which enhances the generation of osteoclasts, whereas the TRPV1 inhibitor (5′-IRTX) reduces osteoclast differentiation. Activation of TRPV1 upregulates tartrate-resistant acid phosphatase activity and the expression of cathepsin K and calcitonin receptor genes, whereas TRPV1 inhibition reverses this effect. The slow release of capsaicin or RTX at a nanomolar concentration from a polysaccharide-based hydrogel enhances bone marrow macrophage (BMM) differentiation into osteoclasts whereas release of 5′-IRTX, an inhibitor of TRPV1, prevents macrophage fusion and osteoclast formation. We also characterize several subcellular parameters, including reactive oxygen (ROS) and nitrogen (RNS) species in the cytosol, mitochondrial, and lysosomal profiles in BMMs. ROS were found to be unaltered upon TRPV1 modulation. NO, however, had elevated levels upon RTX-mediated TRPV1 activation. Capsaicin altered mitochondrial membrane potential (ΔΨm) of BMMs but not 5′-IRTX. Channel modulation had no significant impact on cytosolic pH but significantly altered the pH of lysosomes, making these organelles less acidic. Since BMMs are precursors for osteoclasts, our findings of the cellular physiology of these cells may have broad implications in understanding the role of thermosensitive ion channels in bone formation and functions, and the TRPV1 modulator-releasing hydrogel may have application in bone tissue engineering and other biomedical sectors.

Proteomic Analysis Suggests Altered Mitochondrial Metabolic Profile Associated With Diabetic Cardiomyopathy.

Diabetic cardiomyopathy (DbCM) occurs independently of cardiovascular diseases or hypertension, leading to heart failure and increased risk for death in diabetic patients. To investigate the molecular mechanisms involved in DbCM, we performed a quantitative proteomic profiling analysis in the left ventricle (LV) of type 2 diabetic mice. Six-month-old C57BL/6J-lepr/lepr (db/db) mice exhibited DbCM associated with diastolic dysfunction and cardiac hypertrophy. Using quantitative shotgun proteomic analysis, we identified 53 differentially expressed proteins in the LVs of db/db mice, majorly associated with the regulation of energy metabolism. The subunits of ATP synthase that form the F1 domain, and Cytochrome c1, a catalytic core subunit of the complex III primarily responsible for electron transfer to Cytochrome c, were upregulated in diabetic LVs. Upregulation of these key proteins may represent an adaptive mechanism by diabetic heart, resulting in increased electron transfer and thereby enhancement of mitochondrial ATP production. Conversely, diabetic LVs also showed a decrease in peptide levels of NADH dehydrogenase 1β subcomplex subunit 11, a subunit of complex I that catalyzes the transfer of electrons to ubiquinone. Moreover, the atypical kinase COQ8A, an essential lipid-soluble electron transporter involved in the biosynthesis of ubiquinone, was also downregulated in diabetic LVs. Our study indicates that despite attempts by hearts from diabetic mice to augment mitochondrial ATP energetics, decreased levels of key components of the electron transport chain may contribute to impaired mitochondrial ATP production. Preserved basal mitochondrial respiration along with the markedly reduced maximal respiratory capacity in the LVs of db/db mice corroborate the association between altered mitochondrial metabolic profile and cardiac dysfunction in DbCM.

Exercise reduces intramuscular stress and counteracts muscle weakness in mice with breast cancer.

BackgroundPatients with breast cancer exhibit muscle weakness, which is associated with increased mortality risk and reduced quality of life. Muscle weakness is experienced even in the absence of loss of muscle mass in breast cancer patients, indicating intrinsic muscle dysfunction. Physical activity is correlated with reduced cancer mortality and disease recurrence. However, the molecular processes underlying breast cancer‐induced muscle weakness and the beneficial effect of exercise are largely unknown.MethodsEight‐week‐old breast cancer (MMTV‐PyMT, PyMT) and control (WT) mice had access to active or inactive in‐cage voluntary running wheels for 4 weeks. Mice were also subjected to a treadmill test. Muscle force was measured ex vivo. Tumour markers were determined with immunohistochemistry. Mitochondrial biogenesis and function were assessed with transcriptional analyses of PGC‐1α, the electron transport chain (ETC) and antioxidants superoxide dismutase (Sod) and catalase (Cat), combined with activity measurements of SOD, citrate synthase (CS) and β‐hydroxyacyl‐CoA‐dehydrogenase (βHAD). Serum and intramuscular stress levels were evaluated by enzymatic assays, immunoblotting, and transcriptional analyses of, for example, tumour necrosis factor‐α (TNF‐α) and p38 mitogen‐activated protein kinase (MAPK) signalling.ResultsPyMT mice endured shorter time and distance during the treadmill test (~30%, P < 0.05) and ex vivo force measurements revealed ~25% weaker slow‐twitch soleus muscle (P < 0.001). This was independent of cancer‐induced alteration of muscle size or fibre type. Inflammatory stressors in serum and muscle, including TNF‐α and p38 MAPK, were higher in PyMT than in WT mice (P < 0.05). Cancer‐induced decreases in ETC (P < 0.05, P < 0.01) and antioxidant gene expression were observed (P < 0.05). The exercise intervention counteracted the cancer‐induced muscle weakness and was accompanied by a less aggressive, differentiated tumour phenotype, determined by increased CK8 and reduced CK14 expression (P < 0.05). In PyMT mice, the exercise intervention led to higher CS activity (P = 0.23), enhanced β‐HAD and SOD activities (P < 0.05), and reduced levels of intramuscular stressors together with a normalization of the expression signature of TNFα‐targets and ETC genes (P < 0.05, P < 0.01). At the same time, the exercise‐induced PGC‐1α expression, and CS and β‐HAD activity was blunted in muscle from the PyMT mice as compared with WT mice, indicative that breast cancer interfere with transcriptional programming of mitochondria and that the molecular adaptation to exercise differs between healthy mice and those afflicted by disease.ConclusionsFour‐week voluntary wheel running counteracted muscle weakness in PyMT mice which was accompanied by reduced intrinsic stress and improved mitochondrial and antioxidant profiles and activities that aligned with muscles of healthy mice.

The mitochondrial profile in women with polycystic ovary syndrome: impact of exercise.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder affecting pre-menopausal women and involves metabolic dysregulation. Despite the high prevalence of insulin resistance, the existence of mitochondrial dysregulation and its role in the pathogenesis of PCOS is not clear. Exercise is recommended as the first-line therapy for women with PCOS. In particular, high-intensity interval training (HIIT) is known to improve metabolic health and enhance mitochondrial characteristics. In this narrative review, the existing knowledge of mitochondrial characteristics in skeletal muscle and adipose tissue of women with PCOS and the effect of exercise interventions in ameliorating metabolic and mitochondrial health in these women are discussed. Even though the evidence on mitochondrial dysfunction in PCOS is limited, some studies point to aberrant mitochondrial functions mostly in skeletal muscle, while there is very little research in adipose tissue. Although most exercise intervention studies in PCOS report improvements in metabolic health, they show diverse and inconclusive findings in relation to mitochondrial characteristics. A limitation of the current study is the lack of comprehensive mitochondrial analyses and the diversity in exercise modalities, with only one study investigating the impact of HIIT alone. Therefore, further comprehensive large-scale exercise intervention studies are required to understand the association between metabolic dysfunction and aberrant mitochondrial profile, and the molecular mechanisms underlying the exercise-induced metabolic adaptations in women with PCOS.

Analysis of Mitochondrial Performance in Lymphocytes Using Fluorescent Lifetime Imaging Microscopy.

During lymphocyte maturation and differentiation, cells undergo a series of proliferative stages interrupted with stages of low activity. The rapid proliferation stages are marked by changes in metabolic outputs-adapting to energy demands by either hindering or utilizing metabolic pathways. As such, it is necessary to view these changes in real time; however, current strategies for metabolomics are time consuming and very rarely provide a holistic profile of the cellular metabolism while also characterizing mitochondrial metabolism. Here, we devised a fluorescence lifetime imaging microscopy (FLIM) strategy to image mitochondrial metabolic profiles in lymphocytes as they go through changes in metabolic activity. Our method provides not only a comprehensive view of cellular metabolism but also narrow in mitochondrial contributions while also efficiently excluding non-viable cells with and without the use of a viability dye. Our novel imaging strategy offers a reliable tool to study changes in mitochondrial metabolism.

Multiplexing Seahorse XFe24 and ImageXpress® Nano Platforms for Comprehensive Evaluation of Mitochondrial Bioenergetic Profile and Neuronal Morphology.

The measurement of mitochondrial function has become imperative to understand and characterize diseases characterized by bioenergetic alterations. The advancement of automation and application of high-throughput technologies has propelled ourunderstanding of biological complexity and facilitated drug discovery. Seahorse extracellular flux (XFe) technology measures changes in dissolved oxygen and proton concentration in cell culture media, providing kinetic measurements of oxidative phosphorylation and glycolytic metabolism. ImageXpress® Nano is an automated fluorescent microscope with the ability to perform high-content, fast, and robust imaging in multi-well formats. In this chapter, we present a comprehensive protocol to multiplex the Seahorse XFe24 analyzer withthe ImageXpress® Nano high content imaging microscope to provide a comprehensive yet rigorous profile of bioenergetics and its correlation to neuronal functionand morphology.

Acute exercise dynamically modulates the hepatic mitochondrial proteome.

Exercise powerfully increases energy metabolism and substrate flux in tissues, a process reliant on dramatic changes in mitochondrial energetics. Liver mitochondria play a multi-factorial role during exercise to fuel hepatic glucose output. We previously showed acute exercise activates hepatic mitophagy, a pathway to recycle low-functioning/damaged mitochondria, however little is known how individual bouts of exercise alters the hepatic mitochondrial proteome. Here we leveraged proteomics to examine changes in isolated hepatic mitochondria both immediately after and 2 hours post an acute, 1 hour bout of treadmill exercise in female mice. Further, we utilized leupeptin, a lysosomal inhibitor, to capture and measure exercise-induced changes in mitochondrial proteins that would have been unmeasured due to their targeting for lysosomal degradation. Proteomic analysis of enriched hepatic mitochondria identified 3241 total proteins. Functional enrichment analysis revealed robust enrichment for proteins critical to the mitochondria including metabolic pathways, tricarboxylic acid cycle, and electron transport system. Compared to the sedentary condition, exercise elevated processes regulating lipid localization, Il-5 signaling, and protein phosphorylation in isolated mitochondria. t-SNE analysis identified 4 unique expressional clusters driven by time-dependent changes in protein expression. Isolation of proteins significantly altered with exercise from each cluster revealed influences of leupeptin and exercise both independently and cooperatively modulating mitochondrial protein expressional profiles. Overall, we provide evidence that acute exercise rapidly modulates changes in the proteins/pathways of isolated hepatic mitochondria that include fatty acid metabolism/storage, post-translational protein modification, inflammation, and oxidative stress. In conclusion, the hepatic mitochondrial proteome undergoes extensive remodeling with a bout of exercise.

Mitochondrial Respiration of Platelets: Comparison of Isolation Methods.

Multiple non-aggregatory functions of human platelets (PLT) are widely acknowledged, yet their functional examination is limited mainly due to a lack of standardized isolation and analytic methods. Platelet apheresis (PA) is an established clinical method for PLT isolation aiming at the treatment of bleeding diathesis in severe thrombocytopenia. On the other hand, density gradient centrifugation (DC) is an isolation method applied in research for the analysis of the mitochondrial metabolic profile of oxidative phosphorylation (OXPHOS) in PLT obtained from small samples of human blood. We studied PLT obtained from 29 healthy donors by high-resolution respirometry for comparison of PA and DC isolates. ROUTINE respiration and electron transfer capacity of living PLT isolated by PA were significantly higher than in the DC group, whereas plasma membrane permeabilization resulted in a 57% decrease of succinate oxidation in PA compared to DC. These differences were eliminated after washing the PA platelets with phosphate buffer containing 10 mmol·L−1 ethylene glycol-bis (2-aminoethyl ether)-N,N,N′,N′-tetra-acetic acid, suggesting that several components, particularly Ca2+ and fuel substrates, were carried over into the respiratory assay from the serum in PA. A simple washing step was sufficient to enable functional mitochondrial analysis in subsamples obtained from PA. The combination of the standard clinical PA isolation procedure with PLT quality control and routine mitochondrial OXPHOS diagnostics meets an acute clinical demand in biomedical research of patients suffering from thrombocytopenia and metabolic diseases.

Allopregnanolone potentiates bioenergetic capacity and mitochondrial biogenesis in astrocytes.

We reported previously that the neurosteroid allopregnanolone (Allo) promotes neural stem cell regeneration and differentiation, reverses neurogenic, metabolic and cognitive deficits and reduces Alzheimer's disease (AD) pathology in a mouse model of AD. To further investigate the cell-type specific mechanisms of Allo in regulating brain energy metabolism, we assessed the effect of Allo on mitochondrial bioenergetic profile and biogenesis in rat hippocampal astrocytes. E18 rat hippocampal astrocyte were cultured for 10 days in DMEM:F12(1:1) with 10% FBS and then starved in 10% Charcoal stripped-FBS / DMEM:F12 for 24 hours before treatment with 100nM Allo or 0.001% Vehicle overnight. Upon completion of treatment, cells were subject to morphological, biochemical, metabolic and transcriptomic characterization of their mitochondrial phenotypes. In primary hippocampal astrocytes, Allo significantly attenuates serum deprivation-induced bioenergetic deficits and oxidative stress by enhancing mitochondrial biogenesis and rebalancing mitochondrial dynamics. Allo treatment significantly enhances astrocytic mitochondrial biogenesis via Nrf1/Tfam signaling and reverses mitochondrial hyperfusion by elevating the ratio of mitochondrial fission protein Drp1 to the fusion protein Opa1. Functionally, Allo-induced improvement in bioenergetic function is coupled with reduced inflammasome activation in astrocytes. Outcomes of our findings further support the promising therapeutic effects of Allo against bioenergetic deficits that emerge in early phases of AD, with mitochondria being a major effector.

Extensive mitochondrial proteome disturbance occurs during the early stages of acute myocardial ischemia.

Mitochondrial malfunction leads to the remodeling of myocardial energy metabolism during myocardial ischemia (MI). However, the alterations to the mitochondrial proteome profile during this period has not yet been clarified. An acute MI model was established by high position ligation of the left anterior descending artery in 8-week-old C57BL/6N mice. After 15 min of ligation, the animals were euthanized, and their hearts were collected. The myocardial ultrastructure was observed using transmission electron microscopy (TEM). The cardiac mitochondrial proteome profile was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and bioinformatics analyses. TEM showed that the outer membrane of the mitochondria was dissolved, and the inner membrane (cristae) was corrupted and broken down extensively in the MI group. The mitochondrial membrane potential was decreased. More than 1,700 mitochondrial proteins were identified by LC-MS/MS analysis, and 119 were differentially expressed. Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes functional enrichment analysis showed that endopeptidase activity regulation, the mitochondrial inner membrane, oxidative phosphorylation, the hypoxia-inducible factor-1 signaling pathway, the pentose phosphate pathway and the peroxisome proliferator-activated receptor signaling pathway were involved in the pathophysiological process in the early stage of acute MI. Extensive and substantial changes in the mitochondrial proteins as well as mitochondrial microstructural damage occur in the early stages of acute MI. In the present study, the series of proteins crucially involved in the pathways of mitochondrial dysfunction and metabolism were identified. Further studies are needed to clarify the roles of these proteins in myocardial metabolism remodeling during acute MI injury.

Mitochondriomics reveals the underlying neuroprotective mechanism of TrkB receptor agonist R13 in the 5×FAD mice

Decreased energy metabolism and mitochondrial biogenesis defects are implicated in the pathogenesis of Alzheimer's disease (AD). In present study, mitochondriomics analysis revealed significant effects of R13, a prodrug of 7,8-dihydroxyflavone, on mitochondrial protein expression profile, including the proteins related to the biological processes: fatty acid beta-oxidation, fatty acid metabolic process, mitochondrial electron transport, and mitochondrial respiratory chain. Cluster analysis demonstrated that R13 promoted mitochondrial oxidative phosphorylation (OXPHOS). The functional analysis showed that R13 increased ATP levels and enhanced OXPHOS including complex Ⅰ, Ⅱ, Ⅲ and Ⅳ. R13 treatment increased mitochondrial biogenesis by regulating the levels of p-AMPKα, p-CREB, PGC-1α, NRF1 and TFAM as a consequence of activation of TrkB receptor in the 5 × FAD mice. Finally, R13 significantly reduced the levels of tau phosphorylation and Aβ plaque. Our data suggest that R13 may be used for treating AD via enhancing mitochondrial biogenesis and metabolism.

Abstract 13136: Immune Cell Mitochondrial Function and Diastolic Function in Patients With Heart Failure With Preserved Ejection Fraction

Background: Systemic, multimorbidity-driven inflammation may promote diastolic dysfunction in HFpEF. In this regard, anti-inflammatory therapies have been an attractive treatment strategy in clinical trials of HFpEF. Metabolic reprogramming of immune cells that alter the mitochondrial bioenergetic profile may be an upstream mediator of inflammation. Our aim was to investigate the relationship between the immune cell bioenergetic profile and diastolic function in HFpEF. Methods: Nine female patients with a diagnosis of HFpEF (4 Black, 5 White; Median [IQR]: Age 60 [46-67] yrs; BMI 37 [34-42] kg/m 2 ) were enrolled. The mitochondrial bioenergetic profile was assessed in isolated peripheral blood mononuclear cells by respirometry coupled with modulators of cellular respiration to obtain basal respiration, ATP-linked respiration, maximal respiration and non-mitochondrial respiration. Transthoracic echocardiographic Doppler measures of diastolic function were performed and included mitral inflow velocities (E wave) and early diastolic mitral annular velocity (e’). Results: Diastolic function, represented by E/e’ (Median [IQR]: 10 [8-10] was inversely associated with basal respiration ( r =-0.78, p=0.01), maximal respiration ( r =-0.87, p =0.002), ATP linked respiration (54 [40-105] pmol/min, r =-0.75, p =0.02), non-mitochondrial oxygen consumption (24 [20-35] pmol/min, r =-0.82, p =0.007) and spare respiratory capacity (r=-0.82, p=0.007) as shown in Figure 1. Conclusion: There is a strong negative association between the immune cell mitochondrial bioenergetic profile and diastolic function in HFpEF. Patients with greater impairment in diastolic function, as shown by higher E/e’ ratios, have lower immune cell respiration and reduced respiratory reserve. The relationship between immune cell mitochondrial function, inflammation and diastolic function in HFpEF patients warrants further investigation.

Abstract 12247: Mitochondrial Metabolic Transcriptome Profiles During Cardiomyocyte Differentiation From Mouse and Human Pluripotent Stem Cells

Introduction: Simultaneous increase of myofibrils and mitochondria is a key process of cardiomyocyte differentiation from pluripotent stem cells (PSCs). Specifically, development of mitochondrial oxidative energy metabolism in cardiomyocytes is essential to providing the beating function. Although previous studies reported that mitochondrial oxidative metabolism have some correlation with the differentiation of cardiomyocytes, the mechanism by which mitochondrial oxidative metabolism is regulated and the link between cardiomyogenesis and mitochondrial function are still poorly understood. Methods: We performed transcriptome analysis on cells at specific stages of cardiomyocyte differentiation from mouse embryonic stem cells (mESCs) and human induced PSCs (hiPSCs). We selected highly upregulated mitochondrial metabolic genes at cardiac lineage commitment and time-dependent manner during cardiomyocyte differentiation and identified the protein-protein interaction network connecting between mitochondrial metabolic and cardiac developmental genes. Results: We found several mitochondrial metabolic regulatory genes at cardiac lineage commitment (Cck, Bdnf, Fabp4, Cebpa, Cdkn2a in mESC-derived cells and CCK, NOS3 in hiPSC-derived cells) and time-dependent manner during cardiomyocyte differentiation (Eno3, Pgam2, Cox6a2, Fabp3 in mESC-derived cells and PGAM2, SLC25A4 in hiPSC-derived cells). Notably, mitochondrial metabolic proteins are highly interacted with cardiac developmental proteins time-dependent manner during cardiomyocyte differentiation rather than cardiac lineage commitment. Furthermore, mitochondrial metabolic proteins are mainly interacted with cardiac muscle contractile proteins rather than cardiac transcription factors in cardiomyocyte. Conclusions: Mitochondrial metabolism is critical at cardiac maturation rather than cardiac lineage commitment.

IL-4 Mediated Upregulation of AMPK/SIRT1/PGC-1α Signalling Pathway Is Bypassed By Mitochondrial Complex I Downregulation By NAMPT Inhibition in Venetoclax and Ibrutinib Treated CLL Cells

Introduction: Chronic lymphocytic leukemia (CLL) is one of the most common types of leukemia in adults. Despite significant improvement in the treatment of CLL, drug resistance is emerging when using the single agents ibrutinib or venetoclax. To achieve greater depth of response, combination treatments are being used to eradicate disease. Altered mitochondrial metabolism is a key factor in CLL survival. In order to gain insights into the underlying biology of a promising drug combination treatment, we investigated the combination of venetoclax and ibrutinib on mitochondrial function as well as the B-cell receptor (BCR), apoptotic and adenosine monophosphate activated protein kinase /silent information regulator 1 / peroxisome proliferator-activated receptor-coactivator-1α (AMPK/SIRT1/PGC-1α) signaling pathways in CLL cells. We also evaluated a proposed mechanism of resistance using interleukin-4 (IL-4) to demonstrate the role of a nicotinamide phosphoribosyltransferase (NAMPT) specific inhibitor, FK866, in order to overcome resistance in vitro.Methods: Freshly isolated primary B-cells from CLL patients were treated with venetoclax, ibrutinib or their combination in a dose- and -time responsive fashion. CLL cells were also treated with IL-4 and FK866 in the presence or the absence of the combination treatment. Flow cytometry (Novocyte) was used to assess cell viability, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS). Mitochondrial respiration rates and specific substrate-dependent respiration of individual complexes of the respiratory chain were measured by respirometry (Orobooros O2k oxygraph) and ATP levels by luminometry (Lmax Luminometer, Molecular Devices). Cellular, mitochondrial, and lysosomal morphology was evaluated by Philips CM10 electron microscope and Olympus BX51 fluorescent microscope. Changes in protein levels of signaling pathways were detected by immunoblotting.Results: Each single agent venetoclax or ibrutinib reduced mitochondrial respiration profiles in CLL cells in vitro. The combined effect of these drugs on the respiration profiles, ATP, MMP, ROS and cell viability was more profound than with each agent alone. Proteins involved in 1. BCR [Bruton's tyrosine kinase (BTK); serine/threonine-specific protein kinase (AKT); phospholipase Cɣ2 (PLCɣ2) and extracellular signal-regulated kinase (ERK)], 2. Apoptotic B-cell lymphoma 2 (BCL-2); myeloid cell leukemia-1 (MCL-1) and 3. AMPK/SIRT1/PGC-1α signaling in the venetoclax and ibrutinib combination treated samples were significantly reduced when compared to DMSO and each single agent. AMPK/SIRT1/PGC-1α regulated transcription factors responsible for mitochondrial biogenesis [nuclear respiratory factor (NRF1 and NRF2)] and mitochondrial dynamics related proteins [mitofusin 2 (MFN2) and dynamin-related protein 1 (DRP1)] were preferentially downregulated by the combination treatment. These effects are seen in the morphological changes, as visualized by transmission electron microscopy demonstrating swelling of mitochondria (venetoclax) and vacuole formation (ibrutinib) in addition to the formation of multi-vesicular bodies in the combination. We also validated the impact of the mitochondria and lysosomes using immunofluorescence. In the presence of IL-4 (a secreted cytokine used to activate the BCR), the effects of the combination were negated by the addition of the NAMPT inhibitor, FK866. FK866 also preferentially decreased mitochondrial respiration rates in the presence of Complex I specific substrates and sustained this inhibition in all FK866 containing conditions regardless of IL-4.Conclusions: The combined effect of venetoclax and ibrutinib to target mitochondrial metabolism via the AMPK/SIRT1/PGC-1α signaling pathway provides a rationale for this drug combination treatment. The use of IL-4 identifies a potential path of resistance that can be overcome by NAMPT inhibition by directly targeting Complex I of the electron transport chain of the mitochondria. DisclosuresNo relevant conflicts of interest to declare.