Src-NADH dehydrogenase subunit 2 complex and recognition memory of imprinting in domestic chicks.

Src is a non-receptor tyrosine kinase participating in a range of neuronal processes, including synaptic plasticity. We have recently shown that the amounts of total Src and its two phosphorylated forms, at tyrosine-416 (activated) and tyrosine-527 (inhibited), undergoes time-dependent, region-specific learning-related changes in the domestic chick forebrain after visual imprinting. These changes occur in the intermediate medial mesopallium (IMM), a site of memory formation for visual imprinting, but not the posterior pole of the nidopallium (PPN), a control brain region not involved in imprinting. Src interacts with mitochondrial genome-coded NADH dehydrogenase subunit 2 (NADH2), a component of mitochondrial respiratory complex I. This interaction occurs at brain excitatory synapses bearing NMDA glutamate receptors. The involvement of Src-NADH2 complexes in learning and memory is not yet explored. We show for the first time that, independently of changes in total Src or total NADH2, NADH2 bound to Src immunoprecipitated from the P2 plasma membrane-mitochondrial fraction: (i) is increased in a learning-related manner in the left IMM 1 h after the end of training; (ii), is decreased in the right IMM in a learning-related way 24 h after training. These changes occurred in the IMM but not the PPN. They are attributable to learning occurring during training rather than a predisposition to learn. Learning-related changes in Src-bound NADH2 are thus time- and region-dependent.

Systems genetics analysis defines importance of TMEM43/LUMA for cardiac- and metabolic-related pathways.

Broad cellular functions and diseases including muscular dystrophy, arrhythmogenic right ventricular cardiomyopathy (ARVC5) and cancer are associated with transmembrane protein43 (TMEM43/LUMA). The study aimed to investigate biological roles of TMEM43 through genetic regulation, gene pathways and gene networks, candidate interacting genes, and up- or downstream regulators. Cardiac transcriptomes from 40 strains of recombinant inbred BXD mice and two parental strains representing murine genetic reference population (GRP) were applied for genetic correlation, functional enrichment, and coexpression network analysis using systems genetics approach. The results were validated in a newly created knock-in Tmem43-S358L mutation mouse model (Tmem43S358L) that displayed signs of cardiac dysfunction, resembling ARVC5 phenotype seen in humans. We found high Tmem43 levels among BXDs with broad variability in expression. Expression of Tmem43 highly negatively correlated with heart mass and heart rate among BXDs, whereas levels of Tmem43 highly positively correlated with plasma high-density lipoproteins (HDL). Through finding differentially expressed genes (DEGs) between Tmem43S358L mutant and wild-type (Tmem43WT) lines, 18 pathways (out of 42 found in BXDs GRP) that are involved in ARVC, hypertrophic cardiomyopathy, dilated cardiomyopathy, nonalcoholic fatty liver disease, Alzheimer's disease, Parkinson's disease, and Huntington's disease were verified. We further constructed Tmem43-mediated gene network, in which Ctnna1, Adcy6, Gnas, Ndufs6, and Uqcrc2 were significantly altered in Tmem43S358L mice versus Tmem43WT controls. Our study defined the importance of Tmem43 for cardiac- and metabolism-related pathways, suggesting that cardiovascular disease-relevant risk factors may also increase risk of metabolic and neurodegenerative diseases via TMEM43-mediated pathways.

Deficiency in nebulin repeats of sarcomeric nebulette is detrimental for cardiomyocyte tolerance to exercise and biomechanical stress.

The actin-binding sarcomeric nebulette (NEBL) protein provides efficient contractile flexibility via interaction with desmin intermediate filaments. NEBL gene mutations affecting the nebulin repeat (NR) domain are known to induce cardiomyopathy. The study aimed to explore the roles of NEBL in exercise and biomechanical stress response. We ablated exon3 encoding the first NR of Nebl and created global Neblex3-/ex3- knockout mice. Cardiac function, structure, and transcriptome were assessed before and after a 4-wk treadmill regimen. A Nebl-based exercise signaling network was constructed using systems genetics methods. H9C2 and neonatal rat cardiomyocytes (NRCs) expressing wild-type or mutant NEBL underwent cyclic mechanical strain. Neblex3-/ex3- mice demonstrated diastolic dysfunction with preserved systolic function at 6 mo of age. After treadmill running, 4-mo-old Neblex3-/ex3- mice developed concentric cardiac hypertrophy and left ventricular dilation compared with running Nebl+/+ and sedentary Neblex3-/ex3- mice. Disturbance of sarcomeric Z-disks and thin filaments architecture and disruption of intercalated disks and mitochondria were found in exercised Neblex3-/ex3- mice. A Nebl-based exercise signaling network included Csrp3, Des, Fbox32, Jup, Myh6, and Myh7. Disturbed expression of TM1, DES, JUP, ß-catenin, MLP, α-actinin2, and vinculin proteins was demonstrated. In H9C2 cells, NEBL was recruited into focal adhesions at 24-h poststrain and redistributed along with F-actin at 72-h poststrain, suggesting time-dependent redistribution of NEBL in response to strain. NEBL mutations cause desmin disorganization in NRCs upon stretch. We conclude that Nebl's NR ablation causes disturbed sarcomere, Z-disks, and desmin organization, and prevents NEBL redistribution to focal adhesions in cardiomyocytes, weakening cardiac tolerance to biomechanical stress.NEW & NOTEWORTHY We demonstrate that ablation of first nebulin-repeats of sarcomeric nebulette (Nebl) causes diastolic dysfunction in Neblex3-/ex3- mice. Exercise-induced development of diastolic dysfunction, cardiac hypertrophy and ventricular dilation in knockouts. This was associated with sarcomere disturbance, intercalated disks disruption, and mitochondrial distortion upon stress and altered expression of genes involved in Nebl-based stress network. We demonstrate that G202R and A592 mutations alter actin and desmin expression causing disorganization of desmin filaments upon cyclic strain.

Mitochondrial respiratory supercomplexes in mammalian cells: structural versus functional role.

Mitochondria are recognized as the main source of ATP to meet the energy demands of the cell. ATP production occurs by oxidative phosphorylation when electrons are transported through the electron transport chain (ETC) complexes and develop the proton motive force across the inner mitochondrial membrane that is used for ATP synthesis. Studies since the 1960s have been concentrated on the two models of structural organization of ETC complexes known as "solid-state" and "fluid-state" models. However, advanced new techniques such as blue-native gel electrophoresis, mass spectroscopy, and cryogenic electron microscopy for analysis of macromolecular protein complexes provided new data in favor of the solid-state model. According to this model, individual ETC complexes are assembled into macromolecular structures known as respiratory supercomplexes (SCs). A large number of studies over the last 20 years proposed the potential role of SCs to facilitate substrate channeling, maintain the integrity of individual ETC complexes, reduce electron leakage and production of reactive oxygen species, and prevent excessive and random aggregation of proteins in the inner mitochondrial membrane. However, many other studies have challenged the proposed functional role of SCs. Recently, a third model known as the "plasticity" model was proposed that partly reconciles both "solid-state" and "fluid-state" models. According to the "plasticity" model, respiratory SCs can co-exist with the individual ETC complexes. To date, the physiological role of SCs remains unknown, although several studies using tissue samples of patients or animal/cell models of human diseases revealed an associative link between functional changes and the disintegration of SC assembly. This review summarizes and discusses previous studies on the mechanisms and regulation of SC assembly under physiological and pathological conditions.

Telomeres and Telomerase in Heart Ontogenesis, Aging and Regeneration.

The main purpose of the review article is to assess the contributions of telomere length and telomerase activity to the cardiac function at different stages of development and clarify their role in cardiac disorders. It has been shown that the telomerase complex and telomeres are of great importance in many periods of ontogenesis due to the regulation of the proliferative capacity of heart cells. The review article also discusses the problems of heart regeneration and the identification of possible causes of dysfunction of telomeres and telomerase.

In silico simulation of reversible and irreversible swelling of mitochondria: The role of membrane rigidity.

Mitochondria have been widely accepted as the main source of ATP in the cell. The inner mitochondrial membrane (IMM) is important for the maintenance of ATP production and other functions of mitochondria. The electron transport chain (ETC) generates an electrochemical gradient of protons known as the proton-motive force across the IMM and thus produces the mitochondrial membrane potential that is critical to ATP synthesis. One of the main factors regulating the structural and functional integrity of the IMM is the changes in the matrix volume. Mild (reversible) swelling regulates mitochondrial metabolism and function; however, excessive (irreversible) swelling causes mitochondrial dysfunction and cell death. The central mechanism of mitochondrial swelling includes the opening of non-selective channels known as permeability transition pores (PTPs) in the IMM by high mitochondrial Ca2+ and reactive oxygen species (ROS). The mechanisms of reversible and irreversible mitochondrial swelling and transition between these two states are still unknown. The present study elucidates an upgraded biophysical model of reversible and irreversible mitochondrial swelling dynamics. The model provides a description of the PTP regulation dynamics using an additional differential equation. The rigidity tensor was used in numerical simulations of the mitochondrial parameter dynamics with different initial conditions defined by Ca2+ concentration in the sarco/endoplasmic reticulum. We were able to estimate the values of the IMM rigidity tensor components by fitting the model to the previously reported experimental data. Overall, the model provides a better description of the reversible and irreversible mitochondrial swelling dynamics.

The Mitochondrial Transacylase, Tafazzin, Regulates for AML Stemness by Modulating Intracellular Levels of Phospholipids.

Tafazzin (TAZ) is a mitochondrial transacylase that remodels the mitochondrial cardiolipin into its mature form. Through a CRISPR screen, we identified TAZ as necessary for the growth and viability of acute myeloid leukemia (AML) cells. Genetic inhibition of TAZ reduced stemness and increased differentiation of AML cells both in vitro and in vivo. In contrast, knockdown of TAZ did not impair normal hematopoiesis under basal conditions. Mechanistically, inhibition of TAZ decreased levels of cardiolipin but also altered global levels of intracellular phospholipids, including phosphatidylserine, which controlled AML stemness and differentiation by modulating toll-like receptor (TLR) signaling.

The mitochondrial calcium uniporter underlies metabolic fuel preference in skeletal muscle.

The mitochondrial Ca2+ uniporter (MCU) complex mediates acute mitochondrial Ca2+ influx. In skeletal muscle, MCU links Ca2+ signaling to energy production by directly enhancing the activity of key metabolic enzymes in the mitochondria. Here, we examined the role of MCU in skeletal muscle development and metabolic function by generating mouse models for the targeted deletion of Mcu in embryonic, postnatal, and adult skeletal muscle. Loss of Mcu did not affect muscle growth and maturation or otherwise cause pathology. Skeletal muscle-specific deletion of Mcu in mice also did not affect myofiber intracellular Ca2+ handling, but it did inhibit acute mitochondrial Ca2+ influx and mitochondrial respiration stimulated by Ca2+, resulting in reduced acute exercise performance in mice. However, loss of Mcu also resulted in enhanced muscle performance under conditions of fatigue, with a preferential shift toward fatty acid metabolism, resulting in reduced body fat with aging. Together, these results demonstrate that MCU-mediated mitochondrial Ca2+ regulation underlies skeletal muscle fuel selection at baseline and under enhanced physiological demands, which affects total homeostatic metabolism.

Elucidating the Beneficial Role of PPAR Agonists in Cardiac Diseases.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that bind to DNA and regulate transcription of genes involved in lipid and glucose metabolism. A growing number of studies provide strong evidence that PPARs are the promising pharmacological targets for therapeutic intervention in various diseases including cardiovascular disorders caused by compromised energy metabolism. PPAR agonists have been widely used for decades as lipid-lowering and anti-inflammatory drugs. Existing studies are mainly focused on the anti-atherosclerotic effects of PPAR agonists; however, their role in the maintenance of cellular bioenergetics remains unclear. Recent studies on animal models and patients suggest that PPAR agonists can normalize lipid metabolism by stimulating fatty acid oxidation. These studies indicate the importance of elucidation of PPAR agonists as potential pharmacological agents for protection of the heart from energy deprivation. Here, we summarize and provide a comprehensive analysis of previous studies on the role of PPARs in the heart under normal and pathological conditions. In addition, the review discusses the PPARs as a therapeutic target and the beneficial effects of PPAR agonists, particularly bezafibrate, to attenuate cardiomyopathy and heart failure in patients and animal models.

Author Correction: AMP kinase promotes glioblastoma bioenergetics and tumour growth.

In the version of this Article originally published, in ref. 34 the first author's name was spelled incorrectly. The correct reference is: Rodón, L. et al. Active CREB1 promotes a malignant TGFß2 autocrine loop in glioblastoma. Cancer Discov. 10, 1230-1241 (2014). This has now been amended in all online versions of the Article.

Publisher Correction: AMP kinase promotes glioblastoma bioenergetics and tumour growth.

In the version of this Article originally published, the competing interests statement was missing. The authors declare no competing interests; this statement has now been added in all online versions of the Article.

AMP kinase promotes glioblastoma bioenergetics and tumour growth.

Stress is integral to tumour evolution, and cancer cell survival depends on stress management. We found that cancer-associated stress chronically activates the bioenergetic sensor AMP kinase (AMPK) and, to survive, tumour cells hijack an AMPK-regulated stress response pathway conserved in normal cells. Analysis of The Cancer Genome Atlas data revealed that AMPK isoforms are highly expressed in the lethal human cancer glioblastoma (GBM). We show that AMPK inhibition reduces viability of patient-derived GBM stem cells (GSCs) and tumours. In stressed (exercised) skeletal muscle, AMPK is activated to cooperate with CREB1 (cAMP response element binding protein-1) and promote glucose metabolism. We demonstrate that oncogenic stress chronically activates AMPK in GSCs that coopt the AMPK-CREB1 pathway to coordinate tumour bioenergetics through the transcription factors HIF1α and GABPA. Finally, we show that adult mice tolerate systemic deletion of AMPK, supporting the use of AMPK pharmacological inhibitors in the treatment of GBM.

The Effects of PPAR Stimulation on Cardiac Metabolic Pathways in Barth Syndrome Mice.

Aim: Tafazzin knockdown (TazKD) in mice is widely used to create an experimental model of Barth syndrome (BTHS) that exhibits dilated cardiomyopathy and impaired exercise capacity. Peroxisome proliferator-activated receptors (PPARs) are a group of nuclear receptor proteins that play essential roles as transcription factors in the regulation of carbohydrate, lipid, and protein metabolism. We hypothesized that the activation of PPAR signaling with PPAR agonist bezafibrate (BF) may ameliorate impaired cardiac and skeletal muscle function in TazKD mice. This study examined the effects of BF on cardiac function, exercise capacity, and metabolic status in the heart of TazKD mice. Additionally, we elucidated the impact of PPAR activation on molecular pathways in TazKD hearts. Methods: BF (0.05% w/w) was given to TazKD mice with rodent chow. Cardiac function in wild type-, TazKD-, and BF-treated TazKD mice was evaluated by echocardiography. Exercise capacity was evaluated by exercising mice on the treadmill until exhaustion. The impact of BF on metabolic pathways was evaluated by analyzing the total transcriptome of the heart by RNA sequencing. Results: The uptake of BF during a 4-month period at a clinically relevant dose effectively protected the cardiac left ventricular systolic function in TazKD mice. BF alone did not improve the exercise capacity however, in combination with everyday voluntary running on the running wheel BF significantly ameliorated the impaired exercise capacity in TazKD mice. Analysis of cardiac transcriptome revealed that BF upregulated PPAR downstream target genes involved in a wide spectrum of metabolic (energy and protein) pathways as well as chromatin modification and RNA processing. In addition, the Ostn gene, which encodes the metabolic hormone musclin, is highly induced in TazKD myocardium and human failing hearts, likely as a compensatory response to diminished bioenergetic homeostasis in cardiomyocytes. Conclusion: The PPAR agonist BF at a clinically relevant dose has the therapeutic potential to attenuate cardiac dysfunction, and possibly exercise intolerance in BTHS. The role of musclin in the failing heart should be further investigated.

Loss of SLC25A46 causes neurodegeneration by affecting mitochondrial dynamics and energy production in mice.

Recently, we identified biallelic mutations of SLC25A46 in patients with multiple neuropathies. Functional studies revealed that SLC25A46 may play an important role in mitochondrial dynamics by mediating mitochondrial fission. However, the cellular basis and pathogenic mechanism of the SLC25A46-related neuropathies are not fully understood. Thus, we generated a Slc25a46 knock-out mouse model. Mice lacking SLC25A46 displayed severe ataxia, mainly caused by degeneration of Purkinje cells. Increased numbers of small, unmyelinated and degenerated optic nerves as well as loss of retinal ganglion cells indicated optic atrophy. Compound muscle action potentials in peripheral nerves showed peripheral neuropathy associated with degeneration and demyelination in axons. Mutant cerebellar neurons have large mitochondria, which exhibit abnormal distribution and transport. Biochemically mutant mice showed impaired electron transport chain activity and accumulated autophagy markers. Our results suggest that loss of SLC25A46 causes degeneration in neurons by affecting mitochondrial dynamics and energy production.

Dissection of Z-disc myopalladin gene network involved in the development of restrictive cardiomyopathy using system genetics approach.

AIM: To investigate the regulation of Myopalladin (Mypn) and identify its gene network involved in restrictive cardiomyopathy (RCM). METHODS: Gene expression values were measured in the heart of a large family of BXD recombinant inbred (RI) mice derived from C57BL/6J and DBA/2J. The proteomics data were collected from Mypn knock-in and knock-out mice. Expression quantitative trait locus (eQTL) mapping methods and gene enrichment analysis were used to identify Mypn regulation, gene pathway and co-expression networks. RESULTS: A wide range of variation was found in expression of Mypn among BXD strains. We identified upstream genetic loci at chromosome 1 and 5 that modulate the expression of Mypn. Candidate genes within these loci include Ncoa2, Vcpip1, Sgk3, and Lgi2. We also identified 15 sarcomeric genes interacting with Mypn and constructed the gene network. Two novel members of this network (Syne1 and Myom1) have been confirmed at the protein level. Several members in this network are already known to relate to cardiomyopathy with some novel genes candidates that could be involved in RCM. CONCLUSION: Using systematic genetics approach, we constructed Mypn co-expression networks that define the biological process categories within which similarly regulated genes function. Through this strategy we have found several novel genes that interact with Mypn that may play an important role in the development of RCM.

Mitochondrial permeability transition in cardiac ischemia-reperfusion: whether cyclophilin D is a viable target for cardioprotection?

Growing number of studies provide strong evidence that the mitochondrial permeability transition pore (PTP), a non-selective channel in the inner mitochondrial membrane, is involved in the pathogenesis of cardiac ischemia-reperfusion and can be targeted to attenuate reperfusion-induced damage to the myocardium. The molecular identity of the PTP remains unknown and cyclophilin D is the only protein commonly accepted as a major regulator of the PTP opening. Therefore, cyclophilin D is an attractive target for pharmacological or genetic therapies to reduce ischemia-reperfusion injury in various animal models and humans. Most animal studies demonstrated cardioprotective effects of PTP inhibition; however, a recent large clinical trial conducted by international groups demonstrated that cyclosporine A, a cyclophilin D inhibitor, failed to protect the heart in patients with myocardial infarction. These studies, among others, raise the question of whether cyclophilin D, which plays an important physiological role in the regulation of cell metabolism and mitochondrial bioenergetics, is a viable target for cardioprotection. This review discusses previous studies to provide comprehensive information on the physiological role of cyclophilin D as well as PTP opening in the cell that can be taken into consideration for the development of new PTP inhibitors.

The PPAR pan-agonist bezafibrate ameliorates cardiomyopathy in a mouse model of Barth syndrome.

BACKGROUND: The PGC-1α/PPAR axis has been proposed as a potential therapeutic target for several metabolic disorders. The aim was to evaluate the efficacy of the pan-PPAR agonist, bezafibrate, in tafazzin knockdown mice (TazKD), a mouse model of Barth syndrome that exhibits age-dependent dilated cardiomyopathy with left ventricular (LV) dysfunction. RESULTS: The effect of bezafibrate on cardiac function was evaluated by echocardiography in TazKD mice with or without beta-adrenergic stress. Adrenergic stress by chronic isoproterenol infusion exacerbates the cardiac phenotype in TazKD mice, significantly depressing LV systolic function by 4.5 months of age. Bezafibrate intake over 2 months substantially ameliorates the development of LV systolic dysfunction in isoproterenol-stressed TazKD mice. Without beta-adrenergic stress, TazKD mice develop dilated cardiomyopathy by 7 months of age. Prolonged treatment with suprapharmacological dose of bezafibrate (0.5% in rodent diet) over a 4-month period effectively prevented LV dilation in mice isoproterenol treatment. Bezafibrate increased mitochondrial biogenesis, however also promoted oxidative stress in cardiomyocytes. Surprisingly, improvement of systolic function in bezafibrate-treated mice was accompanied with simultaneous reduction of cardiolipin content and increase of monolysocardiolipin levels in cardiac muscle. CONCLUSIONS: Thus, we demonstrate that bezafibrate has a potent therapeutic effect on preventing cardiac dysfunction in a mouse model of Barth syndrome with obvious implications for treating the human disease. Additional studies are needed to assess the potential benefits of PPAR agonists in humans with Barth syndrome.

Elucidating Mitochondrial Electron Transport Chain Supercomplexes in the Heart During Ischemia-Reperfusion.

AIMS: Mitochondrial supercomplexes (SCs) are the large supramolecular assembly of individual electron transport chain (ETC) complexes that apparently provide highly efficient ATP synthesis and reduce electron leakage and reactive oxygen species (ROS) production. Oxidative stress during cardiac ischemia-reperfusion (IR) can result in degradation of SCs through oxidation of cardiolipin (CL). Also, IR induces calcium overload and enhances reactive oxygen species (mitROS) in mitochondria that result in the opening of the nonselective permeability transition pores (PTP). The opening of the PTP further compromises cellular energetics and increases mitROS ultimately leading to cell death. Here, we examined the role of PTP-induced mitROS in disintegration of SCs during cardiac IR. The relationship between mitochondrial PTP, ROS, and SCs was investigated using Langendorff-perfused rat hearts subjected to global ischemia (25 min) followed by short-time (5 min) or long-time (60 min) reperfusion in the presence or absence of the PTP inhibitor, sanglifehrin A (SfA), and the mitochondrial targeted ROS and electron scavenger, XJB-5-131. Also, the effects of CL deficiency on SC degradation, PTP, and mitROS were investigated in tafazzin knockdown (TazKD) mice. RESULTS: Cardiac IR induced PTP opening and mitROS generation, inhibited by SfA. Percent distributions of SCs were significantly affected by IR, and the effects were dependent on the reperfusion time and reversed by SfA and XJB-5-131. TazKD mice demonstrated a 40% lower SC I + III+IV with reduced basal mitochondrial PTP, ROS, and ETC complex activity. Innovation and Conclusion: Sustained reperfusion after cardiac ischemia induces disintegration of mitochondrial SCs, and PTP-induced ROS presumably play a causal role in SC disassembly. Antioxid. Redox Signal. 27, 57-69.

Creatine transporter deficiency leads to increased whole body and cellular metabolism.

Creatine (Cr) is a guanidino compound required for rapid replenishment of ATP in cells with a high-energy demand. In humans, mutations in the Cr transporter (CRT;SLC6A8) prevent Cr entry into tissue and result in a significant intellectual impairment, epilepsy, and aphasia. The lack of Cr on both the whole body and cellular metabolism was evaluated in Crt knockout (Crt (-/y) ) mice, a high-fidelity model of human CRT deficiency. Crt (-/y) mice have reduced body mass and, however, show a twofold increase in body fat. There was increased energy expenditure in a home cage environment and during treadmill running in Crt (-/y) mice. Consistent with the increases in the whole-body metabolic function, Crt (-/y) mice show increased cellular metabolism as well. Mitochondrial respiration increased in skeletal muscle fibers and hippocampal lysates from Crt (-/y) mice. In addition, Crt (-/y) mice had increased citrate synthase activity, suggesting a higher number of mitochondria instead of an increase in mitochondrial activity. To determine if the increase in respiration was due to increased mitochondrial numbers, we measured oxygen consumption in an equal number of mitochondria from Crt (+/y) and Crt (-/y) mice. There were no changes in mitochondrial respiration when normalized to mitochondrial number, suggesting that the increase in respiration observed could be to higher mitochondrial content in Crt (-/y) mice.