In situ analysis of mitochondrial respiratory capacity - foundation for cellular physiology

Mitochondria are ubiquitous organelles of eukaryotic cells and they are the mayor site of generating energy in the form of adenosine triphoshate through the process of oxidative phosphorylation. Analysis and estimation of mitochondrial function is of outmost importance when it comes to studying intracellular energy metabolism, mechanisms of apoptosis, signaling pathways, calcium storage and the pathophysiology of a large spectrum of human diseases including various neurodegenerative diseases, myopathies, metabolic syndromes and cancer. Respiratory capacity analysis covers one of the many roles that mitochondria play in living cells and it provides us with useful data about functional integrity of mitochondria. Assessment of individual respiratory chain complexes or other mitochondrial enzymes has been widely used to estimate mitochondrial function and dysfunction but it neglects the influence of complex structural and functional interplay of mitochondrial proteins and enzymes and plasmic compounds. Another method that emphasises the importance of studying intact mitochondria is in vitro technique, and although it has many advantages, in some aspects it is far from being representative when it comes to functional assessment of mitochondria. From the perspective of energy production and consumption, the cardiac muscle is a highly demanding tissue and it is the well functioning of mitochondria that is conditio sine qua non for this nature to be fulfilled. In cooperation with the University of Split School of Medicine in Split and under the mentorship of Prof. Marko Ljubkovic, the Department of Physiology of the Faculty of Medicine Novi Sad works on introducing in situ approaches in the analysis of respiratory mitochondrial function in skinned muscle fibers of human cardiac tissue.

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Construction of a mammalian embryo model from stem cells organized by a morphogen signalling centre

Nature Communications ◽

10.1038/s41467-021-23653-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Peng-Fei Xu ◽

Ricardo Moraes Borges ◽

Jonathan Fillatre ◽

Maraysa de Oliveira-Melo ◽

Tao Cheng ◽

...

Keyword(s):

Stem Cells ◽

Cell Tracking ◽

Cardiac Tissue ◽

Embryonic Stem ◽

Mammalian Embryo ◽

Wide Range ◽

Vitro Model ◽

Neurula Stage

AbstractGenerating properly differentiated embryonic structures in vitro from pluripotent stem cells remains a challenge. Here we show that instruction of aggregates of mouse embryonic stem cells with an experimentally engineered morphogen signalling centre, that functions as an organizer, results in the development of embryo-like entities (embryoids). In situ hybridization, immunolabelling, cell tracking and transcriptomic analyses show that these embryoids form the three germ layers through a gastrulation process and that they exhibit a wide range of developmental structures, highly similar to neurula-stage mouse embryos. Embryoids are organized around an axial chordamesoderm, with a dorsal neural plate that displays histological properties similar to the murine embryo neuroepithelium and that folds into a neural tube patterned antero-posteriorly from the posterior midbrain to the tip of the tail. Lateral to the chordamesoderm, embryoids display somitic and intermediate mesoderm, with beating cardiac tissue anteriorly and formation of a vasculature network. Ventrally, embryoids differentiate a primitive gut tube, which is patterned both antero-posteriorly and dorso-ventrally. Altogether, embryoids provide an in vitro model of mammalian embryo that displays extensive development of germ layer derivatives and that promises to be a powerful tool for in vitro studies and disease modelling.

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Important Role of Mitochondria and the Effect of Mood Stabilizers on Mitochondrial Function

Physiological Research ◽

10.33549/physiolres.934324 ◽

2019 ◽

pp. S3-S15 ◽

Cited By ~ 3

Author(s):

M. ĽUPTÁK ◽

J. HROUDOVÁ

Keyword(s):

Mood Disorders ◽

Mitochondrial Function ◽

Krebs Cycle ◽

Cytosolic Calcium ◽

Mood Stabilizers ◽

Calcium Stores ◽

Intermembrane Space ◽

Respiratory Chain Complexes ◽

Atp Formation

Mitochondria primarily serve as source of cellular energy through the Krebs cycle and β-oxidation to generate substrates for oxidative phosphorylation. Redox reactions are used to transfer electrons through a gradient to their final acceptor, oxygen, and to pump hydrogen protons into the intermembrane space. Then, ATP synthase uses the electrochemical gradient to generate adenosine triphosphate (ATP). During these processes, reactive oxygen species (ROS) are generated. ROS are highly reactive molecules with important physiological functions in cellular signaling. Mitochondria play a crucial role in intracellular calcium homeostasis and serve as transient calcium stores. High levels of both, ROS and free cytosolic calcium, can damage mitochondrial and cellular structures and trigger apoptosis. Impaired mitochondrial function has been described in many psychiatric diseases, including mood disorders, in terms of lowered mitochondrial membrane potential, suppressed ATP formation, imbalanced Ca2+ levels and increased ROS levels. In vitro models have indicated that mood stabilizers affect mitochondrial respiratory chain complexes, ROS production, ATP formation, Ca2+ buffering and the antioxidant system. Most studies support the hypothesis that mitochondrial dysfunction is a primary feature of mood disorders. The precise mechanism of action of mood stabilizers remains unknown, but new mitochondrial targets have been proposed for use as mood stabilizers and mitochondrial biomarkers in the evaluation of therapy effectiveness.

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Abstract 30: In vitro Fabrication of Human Cardiac Tissues With Porcine Perfusable Blood Vessels

Circulation Research ◽

10.1161/res.119.suppl_1.30 ◽

2016 ◽

Vol 119 (suppl_1) ◽

Author(s):

Akitoshi Inui ◽

Hidekazu Sekine ◽

Kazunori Sano ◽

Izumi Dobashi ◽

Azumi Yoshida ◽

...

Keyword(s):

Blood Vessels ◽

Cardiac Tissue ◽

Severe Heart Failure ◽

Perfusion Culture ◽

Cardiac Cell ◽

Cell Sheets ◽

Cardiac Tissues ◽

Bioreactor System ◽

Human Cardiac Tissue

The definitive treatment of severe heart failure is heart transplantation; however the number of heart transplantation procedures performed in Japan per year ranges from 30-40 due to donor shortage. Therefore, recently other treatments such as ventricular assist device or regenerative therapy by human cardiac tissue engineering have been developed and are considered as appropriate alternatives. We have developed an original technology, which was named cell-sheet based tissue engineering to fabricate functional three-dimensional tissue by layering cell sheets. The utilization of this technique allowed us to successfully engineer thick rat cardiac tissue with perfusable blood vessels in vitro. Here, we demonstrate a technique to engineer human cardiac tissue with perfusable blood vessels using cardiac cell sheets derived from human induced pluripotent stem cells, and porcine small intestine as a vascular bed for perfusion culture. The small intestine was harvested from with a branch of the superior mesenteric artery and vein and underwent mucosal resection after harvested tissue was cut open. To engineer cardiac tissue with perfusable blood vessels, cardiac cell sheets co-cultured with endothelial cells, were triple-layered and then was overlaid on the vascular bed in the bioreactor system. One day after perfusion culture, overlaid cardiac tissues pulsated spontaneously and were synchronized. The cardiac tissue construct was viable tissue without any observable necrosis. Furthermore we examined the possibility of transplantation of the in vitro engineered human cardiac tissue with the connectable host artery and vein. Engineered cardiac tissue was removed from the bioreactor system after 4-day perfusion, and transplanted to another pig heart. The branch of the superior mesenteric artery and vein of the graft were then reconnected to the host internal thoracic artery and vein. When the cardiac tissue reperfused, it began to beat spontaneously after a few minutes. We believe that this method is useful to fabricate functional cardiac tissue and may become an appropriate treatment for severe heart failure.

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In Vitro Studies of Human Cardiac Tissue

Anesthesiology ◽

10.1097/00000542-197011000-00002 ◽

1970 ◽

Vol 33 (5) ◽

pp. 480-480

Author(s):

RICHARD A. JENSEN

Keyword(s):

Cardiac Tissue ◽

In Vitro Studies ◽

Human Cardiac Tissue

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Inhibiting adenine synthesis attenuates glioblastoma cell stemness and temozolomide resistance

10.1101/2021.06.21.449341 ◽

2021 ◽

Author(s):

Simranjit X. Singh ◽

Rui Yang ◽

Kristen Roso ◽

Landon J. Hansen ◽

Changzheng Du ◽

...

Keyword(s):

Drug Resistance ◽

Mitochondrial Function ◽

Brain Cancer ◽

De Novo ◽

Critical Role ◽

Glioblastoma Cell ◽

Respiratory Capacity ◽

Complementary Approach

Glioblastoma (GBM) is a lethal brain cancer exhibiting high levels of drug resistance, a feature partially imparted by tumor cell stemness. Recent work shows that homozygous MTAP deletion, a genetic alteration occurring in about half of all GBMs, promotes stemness in GBM cells. Exploiting MTAP loss-conferred deficiency in adenine salvage, we demonstrate that transient adenine blockade via treatment with L-Alanosine (ALA), an inhibitor of de novo adenine synthesis, attenuates stemness of MTAP-deficient GBM cells. This ALA-induced reduction in stemness is accompanied by compromised mitochondrial function, highlighted by diminished spare respiratory capacity. Direct pharmacological inhibition of mitochondrial respiration recapitulates the effect of ALA on GBM cell stemness, suggesting ALA targets stemness partially via affecting mitochondrial function. Finally, in agreement with diminished stemness and compromised mitochondrial function, we show that ALA sensitizes GBM cells to temozolomide (TMZ) in vitro and in an orthotopic GBM model. Collectively, these results identify critical roles of adenine supply in maintaining mitochondrial function and stemness of GBM cells, highlight a critical role of mitochondrial function in sustaining GBM stemness, and implicate adenine synthesis inhibition as a complementary approach for treating MTAP-deleted GBMs.

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Modulatory Effects of Alpha- and Gamma-Tocopherol on the Mitochondrial Respiratory Capacity and Membrane Potential in an In Vitro Model of Alzheimer’s Disease

Frontiers in Pharmacology ◽

10.3389/fphar.2021.698833 ◽

2021 ◽

Vol 12 ◽

Author(s):

Aslina Pahrudin Arrozi ◽

Wan Zurinah Wan Ngah ◽

Hanafi Ahmad Damanhuri ◽

Suzana Makpol

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Membrane Potential ◽

Mitochondrial Function ◽

Oxidative Metabolism ◽

Respiratory Capacity ◽

Gamma Tocopherol ◽

Model Of Alzheimer’S Disease ◽

Mitochondrial Oxidative Metabolism

Increased amyloid-beta (Aβ) and amyloid precursor protein (APP) in the brains of Alzheimer’s disease (AD) patients are common pathological hallmarks mediating the disease progression. Growing evidence also suggests that mitochondrial abnormalities are an early feature in the pathogenesis of AD. Intervention with antioxidants has received great interest as a molecular strategy for the manipulation of mitochondrial function. Our previous preliminary study using in vitro cell models expressing different types of APP demonstrated that treatment with alpha-tocopherol (ATF) or gamma-tocopherol (GTF) modulates mitochondrial function by reducing mitochondrial reactive oxygen species (ROS), increasing the production of ATP and preventing apoptosis events, especially in cells expressing the mutant APP form. Thus, we hypothesized that ATF or GTF treatment might also alter mitochondrial metabolic pathways such as oxidative phosphorylation. The present study aimed to investigate the role of ATF and GTF in modulating mitochondrial oxidative metabolism using high-resolution respirometry. Our results showed that both ATF and GTF increased the respiratory capacity and membrane potential in the ROUTINE and OXPHOSCI-LINKED states as well as complex IV enzyme activity in wild-type and mutant APP-overexpressing SH-SY5Y cells. Although preliminary, these findings indicate that ATF and GTF modulate mitochondrial oxidative metabolism in APP-overexpressing cells and, in part, may contribute to the planning of strategies for utilizing vitamin E isomers against mitochondrial-related diseases such as AD.

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A three-dimensional hybrid pacemaker electrode seamlessly integrates into engineered, functional human cardiac tissue in vitro

Scientific Reports ◽

10.1038/s41598-018-32790-8 ◽

2018 ◽

Vol 8 (1) ◽

Cited By ~ 9

Author(s):

Tobias Weigel ◽

Tobias Schmitz ◽

Tobias Pfister ◽

Sabine Gaetzner ◽

Maren Jannasch ◽

...

Keyword(s):

Cardiac Tissue ◽

Three Dimensional ◽

Human Cardiac Tissue

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Mitochondrial Respiratory Capacity is Restored in Hibernating Cardiomyocytes Following Co-Culture with Mesenchymal Stem Cells

Cell Medicine ◽

10.1177/2155179019834938 ◽

2019 ◽

Vol 11 ◽

pp. 215517901983493 ◽

Cited By ~ 3

Author(s):

Laura L. Hocum Stone ◽

Erin Chappuis ◽

Maribel Marquez ◽

Edward O McFalls ◽

Rosemary F. Kelly ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Mitochondrial Function ◽

Cardiac Tissue ◽

Mitochondrial Dynamics ◽

Bypass Graft ◽

Hibernating Myocardium ◽

Large Animal ◽

Respiratory Capacity ◽

Mitochondrial Respiratory

Hibernating myocardium is a subset of ischemic cardiac disease characterized by viable but dysfunctional tissue. Standard treatment for hibernating myocardium is coronary artery bypass graft, which reduces arrhythmias and improves survival but does not fully restore function, presenting a gap in currently available treatments. Large animal studies of hibernating myocardium have identified impaired mitochondrial dynamics as a root cause of persistent cardiac dysfunction despite surgical revascularization. This study presents a novel in vitro model of hibernating myocardium cardiomyocytes to study active mitochondrial respiration in hibernating myocardium cells, and to test the paracrine effect of mesenchymal stem cells on impaired mitochondrial function. Exposure of cardiomyocytes to hypoxic conditions of 1% oxygen for 24 hours resulted in a phenotype consistent with hibernating myocardium cardiac tissue, including decreased respiratory capacity under high work states, decreased expression of mitochondrial proteins, and preserved cellular viability. Co-culture of hibernating myocardium cardiomyocytes with mesenchymal stem cells restored mitochondrial respiratory function, potentially via an increase in proliferator-activated receptor gamma coactivator 1-alpha-driven mitochondrial biogenesis. Co-culture treatment of hibernating myocardium cardiomyocytes with mesenchymal stem cells shows improvement in both mitochondrial function and ATP production, both of which are critical for effectively functioning cardiac tissue. These results suggest that mesenchymal stem cell therapy as an adjunct treatment to revascularization may address the current gap in treatment for hibernating myocardium patients.

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Bioreactor Platform for Biomimetic Culture and in situ Monitoring of the Mechanical Response of in vitro Engineered Models of Cardiac Tissue

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2020.00733 ◽

2020 ◽

Vol 8 ◽

Author(s):

Diana Massai ◽

Giuseppe Pisani ◽

Giuseppe Isu ◽

Andres Rodriguez Ruiz ◽

Giulia Cerino ◽

...

Keyword(s):

Cardiac Tissue ◽

Mechanical Response ◽

In Situ Monitoring

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SARS-CoV-2 infects and induces cytotoxic effects in human cardiomyocytes

10.1101/2020.06.01.127605 ◽

2020 ◽

Cited By ~ 4

Author(s):

Denisa Bojkova ◽

Julian Wagner ◽

Mariana Shumliakivska ◽

Galip Aslan ◽

Umber Saleem ◽

...

Keyword(s):

Cardiac Tissue ◽

Cardiac Injury ◽

Transcriptional Response ◽

Human Cardiomyocytes ◽

Global Pandemic ◽

Virus Rna ◽

Human Cardiac Tissue ◽

Induced Pluripotent ◽

Apoptotic Effects

Background: The coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has emerged as global pandemic. SARS-CoV-2 infection can lead to elevated markers of cardiac injury associated with higher risk of mortality in COVID-19 patients. It is unclear whether cardiac injury may have been caused by direct infection of cardiomyocytes or is mainly secondary to lung injury and inflammation. Here we investigate whether human cardiomyocytes are permissive for SARS-CoV-2 infection. Methods: Infection was induced by two strains of SARS-CoV-2 (FFM1 and FFM2) in human induced pluripotent stem cells-derived cardiomyocytes (hiPS-CM) and in two models of human cardiac tissue. Results: We show that SARS-CoV-2 infects hiPS-CM as demonstrated by detection of intracellular double strand viral RNA and viral spike glycoprotein protein expression. Increasing concentrations of virus RNA are detected in supernatants of infected cardiomyocytes, which induced infections in CaCo-2 cell lines documenting productive infections. SARS-COV-2 infection induced cytotoxic and pro-apoptotic effects and abolished cardiomyocyte beating. RNA sequencing confirmed a transcriptional response to viral infection as demonstrated by the up-regulation of genes associated with pathways related to viral response and interferon signaling, apoptosis and reactive oxygen stress. SARS-CoV-2 infection and cardiotoxicity was confirmed in a iPS-derived human 3D cardiosphere tissue models. Importantly, viral spike protein and viral particles were detected in living human heart slices after infection with SARS-CoV-2. Conclusions: The demonstration that cardiomyocytes are permissive for SARS-CoV-2 infection in vitro warrants the further in depth monitoring of cardiotoxic effects in COVID-19 patients.

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