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Robust Mitochondrial Isolation from Rodent Cardiac Tissue

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry ...

Michigan State University

Oxygen consumption is a key metric of metabolism in aerobic organisms. Current respirometric methods led to seminal discoveries despite limitations such as high sample demand, exchange with atmospheric O, and cumulative titration protocols leading to limited choice of useable tissue, complex data interpretation, and restricted experimental design. We developed a sensitive and customizable method of measuring O consumption rates by a variety of biological samples in microliter volumes without interference from the aerobic environment. We demonstrate that O permeability of the photopolymer, VeroClear, is comparable to that of polyetheretherketone (0.125 . 0.143 barrer, respectively) providing an efficient barrier to oxygen ingress. Optical transparency of VeroClear, combined with high resolution 3D printing, allows for optode-based oxygen detection in enclosed samples. These properties yield a microrespirometer with over 100× dynamic range for O consumption rates. Importantly, the enclosed respirometer configuration and very low oxygen permeability of materials makes it suitable, with resin pre-conditioning, for quantitative assessment of O consumption rates at any desired [O], including hyperbaric, physiological or hypoxic conditions as necessary for each cell type. We characterized two configurations to study soluble enzymes, isolated mitochondria, cells in suspension, and adherent cells cultured on-chip. Improved sensitivity allows for routine quantitative detection of respiration by as few as several hundred cells. Specific activity of cell suspensions in the microrespirometer was in close agreement with that obtained by high-resolution polarographic respirometry. Adherent cell protocols allowed for physiologically relevant assessment of respiration in retinal pigment epithelial cells, ARPE-19, which displayed lower metabolic rates compared with those in suspension. By exchanging medium composition, we demonstrate that cells can be transiently inhibited by cyanide and that 99.6% of basal O uptake is recovered upon its removal. This approach is amenable to new experimental designs and precision measurements on limited sample quantities across basic research and applied fields.

Micro-respirometry of whole cells and isolated mitochondria.

Mitochondrial damage in the cells comprising inner (retinal endothelial cells) and outer (retinal pigment epithelium (RPE)) blood-retinal barriers (BRB) is known to precede the initial BRB breakdown and further histopathological abnormalities in diabetic retinopathy (DR). We previously demonstrated that activation of acid sphingomyelinase (ASM) is an important early event in the pathogenesis of DR, and recent studies have demonstrated that there is an intricate connection between ceramide and mitochondrial function. This study aimed to determine the role of ASM-dependent mitochondrial ceramide accumulation in diabetes-induced RPE cell damage. Mitochondria isolated from streptozotocin (STZ)-induced diabetic rat retinas (7 weeks duration) showed a 1.64 ± 0.29-fold increase in the ceramide-to-sphingomyelin ratio compared to controls. Conversely, the ceramide-to-sphingomyelin ratio was decreased in the mitochondria isolated from ASM-knockout mouse retinas compared to wild-type littermates, confirming the role of ASM in mitochondrial ceramide production. Cellular ceramide was elevated 2.67 ± 1.07-fold in RPE cells derived from diabetic donors compared to control donors, and these changes correlated with increased gene expression of , , and . Treatment of RPE cells derived from control donors with high glucose resulted in elevated , vascular endothelial growth factor (), and intercellular adhesion molecule 1 () mRNA. RPE from diabetic donors showed fragmented mitochondria and a 2.68 ± 0.66-fold decreased respiratory control ratio (RCR). Treatment of immortalized cell in vision research (ARPE-19) cells with high glucose resulted in a 25% ± 1.6% decrease in citrate synthase activity at 72 h. Inhibition of ASM with desipramine (15 μM, 1 h daily) abolished the decreases in metabolic functional parameters. Our results are consistent with diabetes-induced increase in mitochondrial ceramide through an ASM-dependent pathway leading to impaired mitochondrial function in the RPE cells of the retina.

Mitochondrial Ceramide Effects on the Retinal Pigment Epithelium in Diabetes.

Homo sapiens evolved under conditions of intermittent food availability and prolonged fasting between meals. Periods of fasting are important for recovery from meal-induced oxidative and metabolic stress, and tissue repair. Constant high energy-density food availability in present-day society contributes to the pathogenesis of chronic diseases, including diabetes and its complications, with intermittent fasting (IF) and energy restriction shown to improve metabolic health. We have previously demonstrated that IF prevents the development of diabetic retinopathy in a mouse model of type 2 diabetes (db/db); however the mechanisms of fasting-induced health benefits and fasting-induced risks for individuals with diabetes remain largely unknown. Sirtuin 1 (SIRT1), a nutrient-sensing deacetylase, is downregulated in diabetes. In this study, the effect of SIRT1 stimulation by IF, fasting-mimicking cell culture conditions (FMC) or pharmacological treatment using SRT1720 was evaluated on systemic and retinal metabolism, systemic and retinal inflammation and vascular and bone marrow damage.

Fasting and fasting-mimicking treatment activate SIRT1/LXRα and alleviate diabetes-induced systemic and microvascular dysfunction.

Mitochondrial reactive oxygen species (ROS) play important roles in cellular signaling; however, certain pathological conditions such as ischemia/reperfusion (I/R) injury disrupt ROS homeostasis and contribute to cell death. A major impediment to developing therapeutic measures against oxidative stress-induced cellular damage is the lack of a quantitative framework to identify the specific sources and regulatory mechanisms of mitochondrial ROS production. We developed a thermodynamically consistent, mass-and-charge balanced, kinetic model of mitochondrial ROS homeostasis focused on redox sites of electron transport chain complexes I, II, and III. The model was calibrated and corroborated using comprehensive data sets relevant to ROS homeostasis. The model predicts that complex I ROS production dominates other sources under conditions favoring a high membrane potential with elevated nicotinamide adenine dinucleotide (NADH) and ubiquinol (QH) levels. In general, complex I contributes to significant levels of ROS production under pathological conditions, while complexes II and III are responsible for basal levels of ROS production, especially when QH levels are elevated. The model also reveals that hydrogen peroxide production by complex I underlies the non-linear relationship between ROS emission and O at low O concentrations. Lastly, the model highlights the need to quantify scavenging system activity under different conditions to establish a complete picture of mitochondrial ROS homeostasis. In summary, we describe the individual contributions of the electron transport system complex redox sites to total ROS emission in mitochondria respiring under various combinations of NADH- and Q-linked respiratory fuels under varying workloads.

Identifying Site-Specific Superoxide and Hydrogen Peroxide Production Rates From the Mitochondrial Electron Transport System Using a Computational Strategy.

Mitochondrial energy production is crucial for normal daily activities and maintenance of life. Herein, the logic and execution of two main classes of measurements are outlined to delineate mitochondrial function: ATP production and oxygen consumption. Aerobic ATP production is quantified by phosphorus magnetic resonance spectroscopy (PMRS) in vivo in both human subjects and animal models using the same protocols and maintaining the same primary assumptions. Mitochondrial oxygen consumption is quantified by oxygen polarography and applied in isolated mitochondria, cultured cells, and permeabilized fibers derived from human or animal tissue biopsies. Traditionally, mitochondrial functional measures focus on maximal oxidative capacity-a flux rate that is rarely, if ever, observed outside of experimental conditions. Perhaps more physiologically relevant, both measurement classes herein focus on one principal design paradigm; submaximal mitochondrial fluxes generated by graded levels of ADP to map the function for ADP sensitivity. We propose this function defines the bioenergetic role that mitochondria fill within the myoplasm to sense and match ATP demands. Any deficit in this vital role for ATP homeostasis leads to symptoms often seen in cardiovascular and cardiopulmonary diseases, diabetes, and metabolic syndrome.

Yan Levitsky

Department of Physiology

Michigan State University

Robust Mitochondrial Isolation from Rodent Cardiac Tissue

Yan Levitsky

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Physiology

Michigan State University

Robust Mitochondrial Isolation from Rodent Cardiac Tissue

Department of Physiology