scholarly journals Bioenergetic and metabolic impairments in Duchenne Muscular Dystrophy (DMD) patients' iPSC-derived cardiomyocytes

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
L Willi ◽  
B Agranovich ◽  
I Abramovich ◽  
D Freimark ◽  
M Arad ◽  
...  
Keyword(s):  
Stress Test ◽  
Young Male ◽  
Induced Pluripotent Stem Cell ◽  
Dystrophin Gene ◽  
Energy System ◽  
Basal Respiration ◽  
Mitochondrial Activity ◽  
Funding Source ◽  
Atp Production ◽  
Metabolic Impairments

Abstract Introduction DMD, an X-linked muscle degenerative fatal disease, is caused by mutations in the dystrophin gene. Dilated cardiomyopathy (DCM) is a major cause of morbidity and mortality in DMD patients. Treatments for DCM in DMD are limited to steroids and standard heart failure medications such as β-blockers and ACE-inhibitors, and therefore novel therapeutic modalities are urgently needed. Purpose We hypothesized that dystrophin mutations in DMD lead to cardiomyopathy-causing bioenergetic/metabolic impairments, which can be therapeutically targeted for improving cardiac function. Methods Induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CMs) were generated from healthy volunteer and 3 DMD patients: young male (YM), adult male (AM) and adult female (AF). We investigated the bioenergetics, electrophysiology, mitochondrial and metabolic features of healthy and DMD iPSC-CMs using the Seahorse Flux analyzer, patch clamp, confocal fluorescence microscopy and Liquid chromatography mass spectrometry (LC-MS) technologies, respectively. Results To test the hypothesis, we measured respiration and glycolytic rates of healthy and DMD iPSC-CMs. Compared to healthy iPSC-CMs, in both AM and AF DMD, but not in YM DMD cardiomyocytes, there was a 75% decrease in ATP production, and 80% and 45% decrease in basal respiration, respectively. In agreement with the healthy-like bioenergetic status of YM, the iPSC-CMs showed no arrhythmias, in contrast to the prominent arrhythmias in AM and AF cardiomyocytes. To determine whether the impairment in the phosphorylation pathway (OXPHOS) affects glycolysis, we measured the cardiomyocytes' response to glycolytic stress test. These experiments showed that the glycolytic rates were similar in healthy and DMD iPSC-CMs. In agreement with impaired OXPHOS, mitochondrial activity measured by 3D life confocal microscopy was attenuated in the DMD male by 35%, compared to healthy cardiomyocytes. Furthermore, the metabolomic LC-MS analyses demonstrated significant differences in metabolite levels in YM, AM and AF DMD iPSC-CMs relative to healthy iPSC-CMs. For example, compared to healthy iPSC-CMs, there was a dramatic fall to undetected levels in phosphocreatine in both AM and AF, but not in YM DMD, indicating a dysfunctional phosphocreatine energy system. Conclusions DMD iPSC-CMs exhibit bioenergetic/metabolic impairments, which constitute novel targets for alleviating the cardiomyopathy in DMD patients. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): ISF - Israel Science Foundation

Abstract 14072: Bioenergetic and Metabolic Impairments in Duchenne Muscular Dystrophy (DMD) Patients' Induced Pluripotent Stem Cell-derived Cardiomyocytes (iPSC-CMs)

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Lubna Willi ◽  
Jonatan Fernandez-Garcia ◽  
Ifat Abramovich ◽  
Dov Freimark ◽  
Michael Arad ◽  
...  
Keyword(s):  
Young Male ◽  
Induced Pluripotent Stem Cell ◽  
Energy System ◽  
Basal Respiration ◽  
Mitochondrial Activity ◽  
Atp Production ◽  
Therapeutic Modalities ◽  
Ms Analysis ◽  
Induced Pluripotent ◽  
Metabolic Impairments

Introduction: DMD, an X-linked muscle degenerative fatal disease, is caused by mutations in the dystrophin gene. Dilated cardiomyopathy (DCM) is a major cause of morbidity and mortality in DMD patients. Since the DMD DCM treatment is limited, novel therapeutic modalities are urgently needed. Hypothesis: We hypothesized that dystrophin mutations in DMD lead to cardiomyopathy-causing bioenergetic/metabolic impairments, which can be therapeutically targeted for improving cardiac function. Methods: The study included iPSC-CMs from a healthy volunteer and 3 DMD patients (young male, YM; adult male, AM; adult female, AF) and mdx mice. We investigated the bioenergetics, electrophysiology, mitochondrial and metabolic features of iPSC-CMs and heart tissues using the SeaHorse Flux Analyzer, patch clamp, confocal fluorescence microscopy and Liquid Chromatography Mass Spectrometry (LC-MS) technologies, respectively. Results: To test the hypothesis, we measured the iPSC-CM respiration rate using the SeaHorse. Compared to healthy, in both AM and AF DMD, but not in YM DMD cardiomyocytes, ATP production decreased by 75%, and basal respiration decreased by 80% and 45%, respectively. In agreement with impaired oxidative phosphorylation capacity, mitochondrial activity measured by 3D life confocal microscopy was attenuated by 35% in AM, but not in YM. Concurring with the healthy-like bioenergetic status, YM iPSC-CMs fired regularly with no arrhythmias, in contrast to the prominent arrhythmias in AM and AF cardiomyocytes. Further, the LC-MS analysis demonstrated that the levels of 13%, 6% and 7% of the 120 metabolite measured, were lower in AM, AF and YM, while 10%, 18% and 43% were higher, respectively, compared to healthy iPSC-CMs ( p<0.05 ). For example, in AM and AF (but not in YM) the phosphocreatine (PCr) levels were diminished by >80%, compared to healthy iPSC-CMs, indicating a dysfunctional PCr energy system. Finally, the LC-MS analysis of mdx ventricular tissue demonstrated a statistically-significant different metabolic profile in 8 mdx mice vs 8 healthy mice. Conclusion: DMD iPSC-CMs and mdx mice exhibit bioenergetic/metabolic impairments, which constitute novel targets for alleviating the cardiomyopathy in DMD patients.

scholarly journals Palmitate but Not Oleate Exerts a Negative Effect on Oxygen Utilization in Myoblasts of Patients with the m.3243A>G Mutation: A Pilot Study

Life ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 204
Author(s):  
Leila Motlagh Scholle ◽  
Helena Schieffers ◽  
Samiya Al-Robaiy ◽  
Annemarie Thaele ◽  
Diana Lehmann Urban ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Fatty Acids ◽  
Mitochondrial Function ◽  
Stress Responses ◽  
Stress Test ◽  
Saturated Fatty Acids ◽  
Mitochondrial Activity ◽  
Oxygen Utilization ◽  
Atp Production ◽  
Negative Effect

It is known that exposure to excess saturated fatty acids, especially palmitate, can trigger cellular stress responses interpreted as lipotoxicity. The effect of excessive free fatty acids on oxidative phosphorylation capacity in myoblasts of patients with the m.3243A>G mutation was evaluated with the mitochondrial (Mito) stress test using a Seahorse XF96 analyzer. ß-oxidation, measured with the Seahorse XF96 analyzer, was similar in patients and controls, and reduced in both patients and controls at 40 °C compared to 37 °C. Mito stress test in the absence of fatty acids showed lower values in patients compared to controls. The mitochondrial activity and ATP production rates were significantly reduced in presence of palmitate, but not of oleate in patients, showing a negative effect of excessive palmitate on mitochondrial function in patients. Diabetes mellitus is a frequent symptom in patients with m.3243A>G mutation. It can be speculated that the negative effect of palmitate on mitochondrial function might be related to diacylglycerols (DAG) and ceramides (CER) mediated insulin resistance. This might contribute to the elevated risk for diabetes mellitus in m.3243A>G patients.

scholarly journals Cellular and mitochondrial mechanisms of atrial fibrillation

2020 ◽  
Vol 115 (6) ◽  
Author(s):  
Fleur E. Mason ◽  
Julius Ryan D. Pronto ◽  
Khaled Alhussini ◽  
Christoph Maack ◽  
Niels Voigt
Keyword(s):  
Atrial Fibrillation ◽  
Nicotinamide Adenine Dinucleotide ◽  
Molecular Mechanisms ◽  
Treatment Options ◽  
Specific Treatment ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Nicotinamide Adenine ◽  
Mitochondrial Activity ◽  
Atp Production

AbstractThe molecular mechanisms underlying atrial fibrillation (AF), the most common form of arrhythmia, are poorly understood and therefore target-specific treatment options remain an unmet clinical need. Excitation–contraction coupling in cardiac myocytes requires high amounts of adenosine triphosphate (ATP), which is replenished by oxidative phosphorylation in mitochondria. Calcium (Ca2+) is a key regulator of mitochondrial function by stimulating the Krebs cycle, which produces nicotinamide adenine dinucleotide for ATP production at the electron transport chain and nicotinamide adenine dinucleotide phosphate for the elimination of reactive oxygen species (ROS). While it is now well established that mitochondrial dysfunction plays an important role in the pathophysiology of heart failure, this has been less investigated in atrial myocytes in AF. Considering the high prevalence of AF, investigating the role of mitochondria in this disease may guide the path towards new therapeutic targets. In this review, we discuss the importance of mitochondrial Ca2+ handling in regulating ATP production and mitochondrial ROS emission and how alterations, particularly in these aspects of mitochondrial activity, may play a role in AF. In addition to describing research advances, we highlight areas in which further studies are required to elucidate the role of mitochondria in AF.

Abstract P309: Particulate Matter Increases Oxidative Stress And Shortens The Action Potential In IPS-derived Cardiomyocytes

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Mariana Argenziano ◽  
jiajia yang ◽  
Mariana Burgos Angulo ◽  
Thomas V McDonald
Keyword(s):  
Oxidative Stress ◽  
Particulate Matter ◽  
Action Potential ◽  
Cardiac Electrophysiology ◽  
Structural Changes ◽  
Hospital Admissions ◽  
Induced Pluripotent Stem Cell ◽  
Toxic Substances ◽  
Direct Role ◽  
Atp Production

Introduction: Air particulate matter (PM) represents one of the most critical environmental issues worldwide, causing more than 3 million deaths a year. In the US, hospital admissions due to heart failure (HF) increase by 0.8% for every 10 μg/m3 elevation in PM. However, the biological mechanisms behind the effects of PM on cardiovascular disease (CVD) remain poorly defined. Recent studies showed that PM 2.5 can translocate into the circulation, causing cumulative toxicity. With air pollution increasing due to human activity and the growing prevalence of HF, there is a critical need to understand PM's contributions to CVD to develop preventive treatments and novel therapeutic approaches. Hypothesis: We hypothesize that PM can exert its toxic effect by increasing oxidative stress and apoptosis and affecting cardiac electrophysiology. Methods: Three independent induced pluripotent stem cell lines (IPSC) were differentiated into cardiomyocytes (iCMs) and cultured for 30 days before treatment with 100 μg/ml of PM 2.5 for 48h. Experiments including immunostaining, qPCR, RNAseq and Multielectrode Array (MEA) were performed in control (CT) and PM-treated iCMs (PM). Results: Treatment with PM increased ROS and decreased ATP production (CT 9.9±1.2pmol vs PM 6.6±0.8pmol, p<0.01, n=20). Immunostaining showed mitochondrial fragmentation and increased expression of cleaved caspase3 without structural changes. Moreover, PM caused upregulation of the apoptotic markers P53 , PARP1 and CASP3, oxidative stress markers CYP1A1, CYP1B1 and MT2A, and cardiac markers CACNA1C together with downregulation of GJA1 . RNAseq analysis showed upregulation of Gene Ontology terms related to detoxification, response to toxic substances and oxidative stress. Upregulated KEGG pathways included oxidative phosphorylation, hypertrophic cardiomyopathy and dilated cardiomyopathy. MEA experiments revealed a decrease in the spike amplitude and conduction velocity, along with shortening of the action potential (APD90: CT 577±20ms vs. PM 489±16ms, p<0.05, n=20) and increased beat period irregularity (CT 3.2±0.7% vs. PM 13.1±1.6%, p<0.001, n=20). These electrophysiological changes were reversed by treatment with the antioxidant N-acetylcysteine. Conclusions: We conclude that PM plays a direct role in the development of CVD, causing an increase in oxidative stress and affecting the electrophysiology of the heart. Further functional studies in iCMs from HF patients will provide evidence of the effects of these changes on the phenotype of the disease.

Low glucose and high pyruvate reduce the production of 2-oxoaldehydes, improving mitochondrial efficiency, redox regulation and stallion sperm function

2021 ◽  
Author(s):  
J M Ortiz-Rodríguez ◽  
F E Martín-Cano ◽  
G Gaitskell-Phillips ◽  
A Silva ◽  
C Ortega-Ferrusola ◽  
...  
Keyword(s):  
Redox Regulation ◽  
Krebs Cycle ◽  
Tca Cycle ◽  
Mitochondrial Activity ◽  
Atp Production ◽  
Transport Chain ◽  
Low Glucose ◽  
Mitochondrial Efficiency ◽  
Metabolic Strategies ◽  
Go Terms

Abstract Energy metabolism in spermatozoa is complex and involves the metabolism of carbohydrate fatty acids and amino acids. The ATP produced in the electron transport chain (ETC) in the mitochondria appears to be crucial for both sperm motility and maintaining viability, while glycolytic enzymes in the flagella may contribute to ATP production to sustain motility and velocity. Stallion spermatozoa seemingly use diverse metabolic strategies, and in this regard, a study of the metabolic proteome showed that gene ontology (GO) terms and Reactome pathways related to pyruvate metabolism and the Krebs cycle were predominant. Following this, the hypothesis that low glucose concentrations can provide sufficient support for motility and velocity, and thus glucose concentration can be significantly reduced in the medium, was tested. Aliquots of stallion semen in four different media were stored for 48 h at 18°C; a commercial extender containing 67 mM glucose was used as a control. Stallion spermatozoa stored in media with low glucose (1 mM) and high pyruvate (10 mM) (LG-HP) sustained better motility and velocities than those stored in the commercial extender formulated with very high glucose (61.7 ± 1.2% in INRA 96 vs 76.2 ± 1.0% in LG-HP media after 48 h of incubation at 18°C P &lt; 0.0001). Moreover, mitochondrial activity was superior in LG-HP extenders (24.1 ± 1.8% in INRA 96 vs 51.1 ± 0.7% in LG-HP of spermatozoa with active mitochondria after 48 h of storage at 18°C P &lt; 0.0001). Low glucose concentrations may permit more efficient sperm metabolism and redox regulation when substrates for an efficient TCA cycle are provided. The improvement seen using low glucose extenders is due to reductions in the levels of glyoxal and methylglyoxal, 2-oxoaldehydes formed during glycolysis; these compounds are potent electrophiles able to react with proteins, lipids and DNA, causing sperm damage.

scholarly journals Piceatannol Increases Antioxidant Defense and Reduces Cell Death in Human Periodontal Ligament Fibroblast under Oxidative Stress

Antioxidants ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 16 ◽  
Author(s):  
Flávia Póvoa da Costa ◽  
Bruna Puty ◽  
Lygia S. Nogueira ◽  
Geovanni Pereira Mitre ◽  
Sávio Monteiro dos Santos ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Reactive Oxygen Species ◽  
Cell Death ◽  
Cell Viability ◽  
Antioxidant Capacity ◽  
Periodontal Ligament ◽  
Cellular Metabolism ◽  
Mitochondrial Activity ◽  
Oxygen Species ◽  
Atp Production

Piceatannol is a resveratrol metabolite that is considered a potent antioxidant and cytoprotector because of its high capacity to chelate/sequester reactive oxygen species. In pathogenesis of periodontal diseases, the imbalance of reactive oxygen species is closely related to the disorder in the cells and may cause changes in cellular metabolism and mitochondrial activity, which is implicated in oxidative stress status or even in cell death. In this way, this study aimed to evaluate piceatannol as cytoprotector in culture of human periodontal ligament fibroblasts through in vitro analyses of cell viability and oxidative stress parameters after oxidative stress induced as an injury simulator. Fibroblasts were seeded and divided into the following study groups: control, vehicle, control piceatannol, H2O2 exposure, and H2O2 exposure combined with the maintenance in piceatannol ranging from 0.1 to 20 μM. The parameters analyzed following exposure were cell viability by trypan blue exclusion test, general metabolism status by the 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) method, mitochondrial activity through the ATP production, total antioxidant capacity, and reduced gluthatione. Piceatannol was shown to be cytoprotective due the maintenance of cell viability between 1 and 10 μM even in the presence of H2O2. In a concentration of 0.1 μM piceatannol decreased significantly cell viability but increased cellular metabolism and antioxidant capacity of the fibroblasts. On the other hand, the fibroblasts treated with piceatannol at 1 μM presented low metabolism and antioxidant capacity. However, piceatannol did not protect cells from mitochondrial damage as measured by ATP production. In summary, piceatannol is a potent antioxidant in low concentrations with cytoprotective capacity, but it does not prevent all damage caused by hydrogen peroxide.

scholarly journals iPSC-Derived MSCs Versus Originating Jaw Periosteal Cells: Comparison of Resulting Phenotype and Stem Cell Potential

2020 ◽  
Vol 21 (2) ◽  
pp. 587 ◽  
Author(s):  
Felix Umrath ◽  
Marbod Weber ◽  
Siegmar Reinert ◽  
Hans-Peter Wendel ◽  
Meltem Avci-Adali ◽  
...  
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Induced Pluripotent Stem Cell ◽  
Mitochondrial Activity ◽  
Cell Potential ◽  
Differentiation Potential ◽  
Periosteal Cells ◽  
Induced Pluripotent ◽  
Stem Cell Potential ◽  
Promising Source

Induced pluripotent stem cell-derived mesenchymal stem cell-like cells (iMSCs) are considered to be a promising source of progenitor cells for approaches in the field of bone regeneration. In a previous study, we described the generation of footprint-free induced pluripotent stem cells (iPSCs) from human jaw periosteal cells (JPCs) by transfection of a self-replicating RNA (srRNA) and subsequent differentiation into functional osteogenic progenitor cells. In order to facilitate the prospective transfer into clinical practice, xeno-free reprogramming and differentiation methods were established. In this study, we compared the properties and stem cell potential of the iMSCs produced from JPC-derived iPSCs with the parental primary JPCs they were generated from. Our results demonstrated, on the one hand, a comparable differentiation potential of iMSCs and JPCs. Additionally, iMSCs showed significantly longer telomere lengths compared to JPCs indicating rejuvenation of the cells during reprogramming. On the other hand, proliferation, mitochondrial activity, and senescence-associated beta-galactosidase (SA-β-gal) activity indicated early senescence of iMSCs. These data demonstrate the requirement of further optimization strategies to improve mesenchymal development of JPC-derived iPSCs in order to take advantage of the best features of reprogrammed and rejuvenated cells.

Relationships between aerobic and anaerobic energy production in turtle brain in situ

1984 ◽  
Vol 247 (4) ◽  
pp. R740-R744 ◽  
Author(s):  
P. L. Lutz ◽  
P. McMahon ◽  
M. Rosenthal ◽  
T. J. Sick
Keyword(s):  
Ion Homeostasis ◽  
Creatine Phosphate ◽  
High Energy ◽  
Mammalian Brain ◽  
Mitochondrial Activity ◽  
Atp Production ◽  
Anaerobic Energy ◽  
Complete Failure ◽  
Lactate Levels

To define the relationships between brain mitochondrial activity and high-energy intermediates in the turtle, brains were frozen in situ at times determined by the reduction-oxidation status of cytochrome a,a3. Increases in brain lactate indicated that transition to anaerobiosis occurred while oxidative reactions were still ongoing during hypoxia and that the signal for anaerobiosis is not the complete failure of mitochondrial electron transfer. Decreases in creatine phosphate (CrP), ATP, ADP, and AMP demonstrate that their consumption exceeded production during hypoxia. CrP decreased further during the following 120 min of anoxia, but ATP, ADP, and AMP were not significantly altered from control values. These data suggest that ATP is not inhibitory to rate-limiting, key glycolytic enzymes such as phosphofructokinase. Calculations indicate that ATP production declined during anoxia. This study indicates that decreased CrP values, decreased ATP production, and high lactate levels do not preclude ion homeostasis and electrical activity of turtle brain and that this brain is protected against lactate or pH damage during anoxia, in contrast to mammalian brain.

Bioenergetics and mitochondrial transmembrane potential during differentiation of cultured osteoblasts

2000 ◽  
Vol 279 (4) ◽  
pp. C1220-C1229 ◽  
Author(s):  
Svetlana V. Komarova ◽  
Fasoil I. Ataullakhanov ◽  
Ruth K. Globus
Keyword(s):  
Transmembrane Potential ◽  
Lactate Production ◽  
Glucose Consumption ◽  
Confocal Imaging ◽  
Metabolic Inhibitors ◽  
Mitochondrial Activity ◽  
Atp Production ◽  
Fourfold Increase ◽  
Fetal Rat ◽  
Immature Cells

To evaluate the relationship between osteoblast differentiation and bioenergetics, cultured primary osteoblasts from fetal rat calvaria were grown in medium supplemented with ascorbate to induce differentiation. Before ascorbate treatment, the rate of glucose consumption was 320 nmol · h−1· 106cells−1, respiration was 40 nmol · h−1· 106cells−1, and the ratio of lactate production to glucose consumption was ∼2, indicating that glycolysis was the main energy source for immature osteoblasts. Ascorbate treatment for 14 days led to a fourfold increase in respiration, a threefold increase in ATP production, and a fivefold increase in ATP content compared with that shown in immature cells. Confocal imaging of mitochondria stained with a transmembrane potential-sensitive vital dye showed that mature cells possessed abundant amounts of high-transmembrane-potential mitochondria, which were concentrated near the culture medium-facing surface. Acute treatment of mature osteoblasts with metabolic inhibitors showed that the rate of glycolysis rose to maintain the cellular energy supply constant. Thus progressive differentiation coincided with changes in cellular metabolism and mitochondrial activity, which are likely to play key roles in osteoblast function.

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