γ-Rays-generated ROS induce apoptosis via mitochondrial and cell cycle alteration in smooth muscle cells

Abstract

Purpose: γ-rays (IR) cause an increase in intracellular calcium [Ca²⁺], alters contractility and triggers apoptosis via the activation of protein kinase C in intestinal guinea pig smooth muscle cells. The present study investigated the role of the mitochondria in these processes and characterized proteins involved in IR-induced apoptosis.

Materials and methods: Intestinal smooth muscle cells were exposed to 10–50 Gy from a ⁶⁰Co γ-source. Reactive oxygen species (ROS) levels were measured by colourimetry with a fluorescente probe. Protein expression was analyzed by immunoblotting and immunofluorescence.

Results: Apoptosis was inhibited by glutathione, possible by inhibiting the generation or scavenging ROS. Apoptosis was mediated by the mitochondria releasing cytochrome c leading to caspase 3 activation. IR increased the expression of the cyclins A, B2 and E and led to unbalanced cellular growth in an absorption dose-dependent manner. However, radiation did not induce alterations in the mitochondrial ultrastructure or in transmembrane electric potential. In contrast, IR increased the nuclear expression of cytoplasmic proteins and cyclins A and E.

Conclusion: Smooth muscle cells subjected to IR undergo mitochondrial-mediated apoptosis that involves oncoproteins activation and preserves mitochondrial structure. IR also cause alterations in the expression and localization of both pro- and anti-apoptotic proteins.     

TAp73 depletion accelerates aging through metabolic dysregulation

Aging is associated with impaired scavenging of reactive oxygen species (ROS). Here, we show that TAp73, a p53 family member, protects against aging by regulating mitochondrial activity and preventing ROS accumulation. TAp73-null mice show more pronounced aging with increased oxidative damage and senescence. TAp73 deletion reduces cellular ATP levels, oxygen consumption, and mitochondrial complex IV activity, with increased ROS production and oxidative stress sensitivity. We show that the mitochondrial complex IV subunit cytochrome C oxidase subunit 4 (Cox4i1) is a direct TAp73 target and that Cox4i1 knockdown phenocopies the cellular senescence of TAp73-null cells. Results indicate that TAp73 affects mitochondrial respiration and ROS homeostasis, thus regulating aging.     

Melatonin enhances neural stem cell differentiation and engraftment by increasing mitochondrial function

Neural stem cells (NSCs) are regarded as a promising therapeutic approach to protecting and restoring damaged neurons in neurodegenerative diseases (NDs) such as Parkinson's disease and Alzheimer's disease (PD and AD, respectively). However, new research suggests that NSC differentiation is required to make this strategy effective. Several studies have demonstrated that melatonin increases mature neuronal markers, which reflects NSC differentiation into neurons. Nevertheless, the possible involvement of mitochondria in the effects of melatonin during NSC differentiation has not yet been fully established. We therefore tested the impact of melatonin on NSC proliferation and differentiation in an attempt to determine whether these actions depend on modulating mitochondrial activity. We measured proliferation and differentiation markers, mitochondrial structural and functional parameters as well as oxidative stress indicators and also evaluated cell transplant engraftment. This enabled us to show that melatonin (25 μM) induces NSC differentiation into oligodendrocytes and neurons. These effects depend on increased mitochondrial mass/DNA/complexes, mitochondrial respiration, and membrane potential as well as ATP synthesis in NSCs. It is also interesting to note that melatonin prevented oxidative stress caused by high levels of mitochondrial activity. Finally, we found that melatonin enriches NSC engraftment in the ND mouse model following transplantation. We concluded that a combined therapy involving transplantation of NSCs pretreated with pharmacological doses of melatonin could efficiently restore neuronal cell populations in PD and AD mouse models depending on mitochondrial activity promotion.     

Systems biology of lupus: Mapping the impact of genomic and environmental factors on gene expression signatures,cellular signaling,metabolic pathways,hormonal and cytokine imbalance,and selecting targets for treatment

Systemic lupus erythematosus (SLE) is characterized by the dysfunction of T cells, B cells, and dendritic cells, the release of pro-inflammatory nuclear materials from necrotic cells, and the formation of antinuclear antibodies (ANA) and immune complexes of ANA with DNA, RNA, and nuclear proteins. Activation of the mammalian target of rapamycin (mTOR) has recently emerged as a key factor in abnormal activation of T and B cells in SLE. In T cells, increased production of nitric oxide and mitochondrial hyperpolarization (MHP) were identified as metabolic checkpoints upstream of mTOR activation. mTOR controls the expression T-cell receptor-associated signaling proteins CD4 and CD3ζ through increased expression of the endosome recycling regulator Rab5 and HRES-1/Rab4 genes, enhances Ca²⁺ fluxing and skews the expression of tyrosine kinases both in T and B cells, and blocks the expression of Foxp3 and the generation of regulatory T cells. MHP, increased activity of mTOR, Rab GTPases, and Syk kinases, and enhanced Ca²⁺ flux have emerged as common T and B cell biomarkers and targets for treatment in SLE.     

Melatonin attenuates postmyocardial infarction injury via increasing Tom70 expression

Mitochondrial dysfunction leads to reactive oxygen species (ROS) overload, exacerbating injury in myocardial infarction (MI). As a receptor for translocases in the outer mitochondrial membrane (Tom) complex, Tom70 has an unknown function in MI, including melatonin‐induced protection against MI injury. We delivered specific small interfering RNAs against Tom70 or lentivirus vectors carrying Tom70a sequences into the left ventricles of mice or to cultured neonatal murine ventricular myocytes (NMVMs). At 48 h post‐transfection, the left anterior descending coronary arteries of mice were permanently ligated, while the NMVMs underwent continuous hypoxia. At 24 h after ischemia/hypoxia, oxidative stress was assessed by dihydroethidium and lucigenin‐enhanced luminescence, mitochondrial damage by transmission electron microscopy and ATP content, and cell apoptosis by terminal deoxynucleotidyl transferase dUTP nick‐end labeling and caspase‐3 assay. At 4 weeks after ischemia, cardiac function and fibrosis were evaluated in mice by echocardiography and Masson's trichrome staining, respectively. Ischemic/hypoxic insult reduced Tom70 expression in cardiomyocytes. Tom70 downregulation aggravated post‐MI injury, with increased mitochondrial fragmentation and ROS overload. In contrast, Tom70 upregulation alleviated post‐MI injury, with improved mitochondrial integrity and decreased ROS production. PGC‐1α/Tom70 expression in ischemic myocardium was increased with melatonin alone, but not when combined with luzindole. Melatonin attenuated post‐MI injury in control but not in Tom70‐deficient mice. N‐acetylcysteine (NAC) reversed the adverse effects of Tom70 deficiency in mitochondria and cardiomyocytes, but at a much higher concentration than melatonin. Our findings showed that Tom70 is essential for melatonin‐induced protection against post‐MI injury, by breaking the cycle of mitochondrial impairment and ROS generation.     

Melatonin regulates lipid metabolism in porcine oocytes

It is being increasingly recognized that the processes of lipogenesis and lipolysis are important for providing an essential energy source during oocyte maturation and embryo development. Recent studies demonstrated that melatonin has a role in lipid metabolism regulation, including lipogenesis, lipolysis, and mitochondrial biogenesis. In this study, we attempted to investigate the effects of melatonin on lipid metabolism during porcine oocyte in vitro maturation. Melatonin treatment significantly enhanced the number of lipid droplets (LDs) and upregulated gene expression related to lipogenesis (ACACA, FASN, PPARγ, and SREBF1). Oocytes treated with melatonin formed smaller LDs and abundantly expressed several genes associated with lipolysis, including ATGL, CGI‐58, HSL, and PLIN2. Moreover, melatonin significantly increased the content of fatty acids, mitochondria, and ATP, as indicated by fluorescent staining. Concomitantly, melatonin treatment upregulated gene expression related to fatty acid β‐oxidation (CPT1a, CPT1b, CPT2, and ACADS) and mitochondrial biogenesis (PGC‐1α, TFAM, and PRDX2). Overall, melatonin treatment not only altered both the morphology and amount of LDs, but also increased the content of fatty acids, mitochondria, and ATP. In addition, melatonin upregulated mRNA expression levels of lipogenesis, lipolysis, β‐oxidation, and mitochondrial biogenesis‐related genes in porcine oocytes. These results indicated that melatonin promoted lipid metabolism and thereby provided an essential energy source for oocyte maturation and subsequent embryonic development.     

Newly-found functions of metformin for the prevention and treatment of age-related macular degeneration

AIM: To explore the temporal mitochondrial characteristics of retinal pigment epithelium (RPE) cells obtained from human embryonic stem cells (hESC)-derived retinal organoids (hEROs-RPE), to verify the optimal period for using hEROs-RPE as donor cells from the aspect of mitochondria and to optimize RPE cell-based therapeutic strategies for age-related macular degeneration (AMD). METHODS: RPE cells were obtained from hEROs and from spontaneous differentiation (SD-RPE). The mitochondrial characteristics were analyzed every 20d from day 60 to 160. Mitochondrial quantity was measured by MitoTracker Green staining. Transmission electron microscopy was adopted to assess the morphological features of the mitochondria, including their distribution, length, and cristae. Mitochondrial membrane potentials (MMPs) were determined by JC-1 staining and flow cytometry. ROS levels were evaluated by flow cytometry, and ATP levels were measured by a luminometer. Differences between two groups were analyzed by the independent-samples t-test, and comparisons among multiple groups were made using one-way ANOVA or Kruskal-Wallis H test when equal variance is not assumed. RESULTS: hEROs-RPE and SD-RPE cells from day 60 to 160 were successfully differentiated from hESCs and expressed RPE-specific markers (Pax6, mitf, Bestrophin-1, RPE65, Cralbp). RPE features, including a cobblestone-like morphology with tight junctions (ZO-1), pigments and microvilli, were also observed in both hERO-RPE and SD-RPE cells. The mitochondrial quantities peaked in both hEROs-RPE and SD-RPE cells at day 80. However, the cristae of hEROs mitochondria were less mature and abundant than those of SD mitochondria at day 80, with hEROs mitochondria becoming mature at day 100. Both hEROs-RPE and SD-RPE cells showed low ROS levels from day 100 to 140 and maintained a normal MMP during this period. However, hEROs mitochondria maintained a longer time to produce high levels of ATP (from day 120 to 140) than SD-RPE cells (only day 120). CONCLUSION: Mitochondria of hEROs-RPE cells develop slower and maintain a longer time to supply high-levels of energy than SD-RPE cells. From a mitochondrial aspect, hEROs-RPE cells from day 100 to 140 are an optimal cell source for treating AMD.     

Altered Fetal Skeletal Muscle Nutrient Metabolism Following an Adverse In Utero Environment and the Modulation of Later Life Insulin Sensitivity

The importance of the in utero environment as a contributor to later life metabolic disease has been demonstrated in both human and animal studies. In this review, we consider how disruption of normal fetal growth may impact skeletal muscle metabolic development, ultimately leading to insulin resistance and decreased insulin sensitivity, a key precursor to later life metabolic disease. In cases of intrauterine growth restriction (IUGR) associated with hypoxia, where the fetus fails to reach its full growth potential, low birth weight (LBW) is often the outcome, and early in postnatal life, LBW individuals display modifications in the insulin-signaling pathway, a critical precursor to insulin resistance. In this review, we will present literature detailing the classical development of insulin resistance in IUGR, but also discuss how this impaired development, when challenged with a postnatal Western diet, may potentially contribute to the development of later life insulin resistance. Considering the important role of the skeletal muscle in insulin resistance pathogenesis, understanding the in utero programmed origins of skeletal muscle deficiencies in insulin sensitivity and how they may interact with an adverse postnatal environment, is an important step in highlighting potential therapeutic options for LBW offspring born of pregnancies characterized by placental insufficiency.     

Increasing mitochondrial ATP synthesis with butyrate normalizes ADP and contractile function in metabolic heart disease

Metabolic heart disease (MHD), which is strongly associated with heart failure with preserved ejection fraction, is characterized by reduced mitochondrial energy production and contractile performance. In this study, we tested the hypothesis that an acute increase in ATP synthesis, via short chain fatty acid (butyrate) perfusion, restores contractile function in MHD. Isolated hearts of mice with MHD due to consumption of a high fat high sucrose (HFHS) diet or on a control diet (CD) for 4 months were studied using ³¹P NMR spectroscopy to measure high energy phosphates and ATP synthesis rates during increased work demand. At baseline, HFHS hearts had increased ADP and decreased free energy of ATP hydrolysis (ΔG _{~ ATP}), although contractile function was similar between the two groups. At high work demand, the ATP synthesis rate in HFHS hearts was reduced by over 50%. Unlike CD hearts, HFHS hearts did not increase contractile function at high work demand, indicating a lack of contractile reserve. However, acutely supplementing HFHS hearts with 4mM butyrate normalized ATP synthesis, ADP, ΔG _{~ ATP} and contractile reserve. Thus, acute reversal of depressed mitochondrial ATP production improves contractile dysfunction in MHD. These findings suggest that energy starvation may be a reversible cause of myocardial dysfunction in MHD, and opens new therapeutic opportunities.