Estrogen maintains mitochondrial content and function in the right ventricle of rats with pulmonary hypertension

The typical cause of death in pulmonary hypertension (PH) is right ventricular (RV) failure, with females showing better survival rates than males. Recently, metabolic shift and mitochondrial dysfunction have been demonstrated in RV failure secondary to PH. In light of evidence showing that estrogen protects mitochondrial function and biogenesis in noncardiovascular systems, we hypothesized that the mechanism by which estrogen preserves RV function is via protection of mitochondrial content and oxidative capacity in PH. We used a well‐established model of PH (Sugen+Hypoxia) in ovariectomized female rats with/without estrogen treatment. RV functional measures were derived from pressure–volume relationships measured via RV catheterization in live rats. Citrate synthase activity, a marker of mitochondrial density, was measured in both RV and LV tissues. Respiratory capacity of mitochondria isolated from RV was measured using oxygraphy. We found that RV ventricular‐vascular coupling efficiency decreased in the placebo‐treated SuHx rats (0.78 ± 0.10 vs. 1.50 ± 0.13 in control, P < 0.05), whereas estrogen restored it. Mitochondrial density decreased in placebo‐treated SuHx rats (0.12 ± 0.01 vs. 0.15 ± 0.01 U citrate synthase/mg in control, P < 0.05), and estrogen attenuated the decrease. Mitochondrial quality and oxidative capacity tended to be lower in placebo‐treated SuHx rats only. The changes in mitochondrial biogenesis and function paralleled the expression levels of PGC‐1α in RV. Our results suggest that estrogen protects RV function by preserving mitochondrial content and oxidative capacity. This provides a mechanism by which estrogen provides protection in female PH patients and paves the way to develop estrogen and its targets as a novel RV‐specific therapy for PH.Motivated by the clinical observation that female patients have superior right ventricular (RV) functional adaption in pulmonary hypertension (PH), we investigated the metabolic mechanisms by which estrogen offers protection to RV function in a rat model of PH. Our study for the first time reveals that estrogen preserves RV mitochondrial density and tends to preserve function in PH‐induced RV hypertrophy, which may underlie the estrogenic improvement of RV contractility and mechanical efficiency. We believe that our findings will contribute to an improved understanding of RV failure and more effective therapies to combat this devastating disease.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/136542/1/phy213157.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/136542/2/phy213157_am.pd

Analysis of Diffusion of Ras2 in Saccharomyces cerevisiae Using Fluorescence Recovery after Photobleaching

Binding, lateral diffusion and exchange are fundamental dynamic processes involved in protein association with cellular membranes. In this study, we developed numerical simulations of lateral diffusion and exchange of fluorophores in membranes with arbitrary bleach geometry and exchange of the membrane localized fluorophore with the cytosol during Fluorescence Recovery after Photobleaching (FRAP) experiments. The model simulations were used to design FRAP experiments with varying bleach region sizes on plasma-membrane localized wild type GFP-Ras2 with a dual lipid anchor and mutant GFP-Ras2C318S with a single lipid anchor in live yeast cells to investigate diffusional mobility and the presence of any exchange processes operating in the time scale of our experiments. Model parameters estimated using data from FRAP experiments with a 1 micron x 1 micron bleach region-of-interest (ROI) and a 0.5 micron x 0.5 micron bleach ROI showed that GFP-Ras2, single or dual lipid modified, diffuses as single species with no evidence of exchange with a cytoplasmic pool. This is the first report of Ras2 mobility in yeast plasma membrane. The methods developed in this study are generally applicable for studying diffusion and exchange of membrane associated fluorophores using FRAP on commercial confocal laser scanning microscopes.Comment: Accepted for publication in Physical Biology (2010). 28 pages, 7 figures, 3 table

Mitochondrial structure and function are not different between nonfailing donor and end‐stage failing human hearts

During human heart failure, the balance of cardiac energy use switches from predominantly fatty acids (FAs) to glucose. We hypothesized that this substrate shift was the result of mitochondrial degeneration; therefore, we examined mitochondrial oxidation and ultrastructure in the failing human heart by using respirometry, transmission electron microscopy, and gene expression studies of demographically matched donor and failing human heart left ventricular (LV) tissues. Surprisingly, respiratory capacities for failing LV isolated mitochondria (n = 9) were not significantly diminished compared with donor LV isolated mitochondria (n = 7) for glycolysis (pyruvate + malate)‐ or FA (palmitoylcarnitine)‐derived substrates, and mitochondrial densities, assessed via citrate synthase activity, were consistent between groups. Transmission electron microscopy images also showed no ultrastructural remodeling for failing vs. donor mitochondria; however, the fraction of lipid droplets (LDs) in direct contact with a mitochondrion was reduced, and the average distance between an LD and its nearest neighboring mitochondrion was increased. Analysis of FA processing gene expression between donor and failing LVs revealed 0.64‐fold reduced transcript levels for the mitochondrial‐LD tether, perilipin 5, in the failing myocardium (P = 0.003). Thus, reduced FA use in heart failure may result from improper delivery, potentially via decreased perilipin 5 expression and mitochondrial‐LD tethering, and not from intrinsic mitochondrial dysfunction.—Holzem, K. M., Vinnakota, K. C., Ravikumar, V. K., Madden, E. J., Ewald, G. A., Dikranian, K., Beard, D. A., Efimov, I. R. Mitochondrial structure and function are not different between nonfailing donor and end‐stage failing human hearts. FASEB J. 30, 2698‐2707 (2016). www.fasebj.orgPeer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154292/1/fsb2fj201500118r.pd

Network Modeling of Liver Metabolism to Predict Plasma Metabolite Changes During Short-Term Fasting in the Laboratory Rat

The liver—a central metabolic organ that integrates whole-body metabolism to maintain glucose and fatty-acid regulation, and detoxify ammonia—is susceptible to injuries induced by drugs and toxic substances. Although plasma metabolite profiles are increasingly investigated for their potential to detect liver injury earlier than current clinical markers, their utility may be compromised because such profiles are affected by the nutritional state and the physiological state of the animal, and by contributions from extrahepatic sources. To tease apart the contributions of liver and non-liver sources to alterations in plasma metabolite profiles, here we sought to computationally isolate the plasma metabolite changes originating in the liver during short-term fasting. We used a constraint-based metabolic modeling approach to integrate central carbon fluxes measured in our study, and physiological flux boundary conditions gathered from the literature, into a genome-scale model of rat liver metabolism. We then measured plasma metabolite profiles in rats fasted for 5–7 or 10–13 h to test our model predictions. Our computational model accounted for two-thirds of the observed directions of change (an increase or decrease) in plasma metabolites, indicating their origin in the liver. Specifically, our work suggests that changes in plasma lipid metabolites, which are reliably predicted by our liver metabolism model, are key features of short-term fasting. Our approach provides a mechanistic model for identifying plasma metabolite changes originating in the liver

Detailed Enzyme Kinetics in Terms of Biochemical Species: Study of Citrate Synthase

The compulsory-ordered ternary catalytic mechanism for two-substrate two-product enzymes is analyzed to account for binding of inhibitors to each of the four enzyme states and to maintain the relationship between the kinetic constants and the reaction equilibrium constant. The developed quasi-steady flux expression is applied to the analysis of data from citrate synthase to determine and parameterize a kinetic scheme in terms of biochemical species, in which the effects of pH, ionic strength, and cation binding to biochemical species are explicitly accounted for in the analysis of the data. This analysis provides a mechanistic model that is consistent with the data that have been used support competing hypotheses regarding the catalytic mechanism of this enzyme

Analysis using random-order model of Equation (26).

<p>Data and conditions in A, B, C, and D are the same as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001825#pone-0001825-g002" target="_blank">Figure 2</a>. Data and conditions for E and F are the same as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001825#pone-0001825-g003" target="_blank">Figures 3A and 3B</a>, respectively Parameter values for solid line model predictions are <i>V_max</i> = 0.320 µmol·min⁻¹· µg⁻¹, <i>K_mB</i> = 6.20 µM, <i>K_mP</i> = 8.00 µM, <i>K_eA</i> = 1.35 µM, <i>K_eB</i> = 1.10 µM, <i>K_eP</i> = 21.6 nM, <i>K_eQ</i> = 0.150 µM. Parameter values for dashed line model predictions are <i>V_max</i> = 0.526 µmol·min⁻¹·µg⁻¹, <i>K_mB</i> = 36.6 µM, <i>K_mP</i> = 80.792 mM, <i>K_eA</i> = 3.08 nM, <i>K_eB</i> = 10.8 nM, <i>K_eP</i> = 0.152 µM, <i>K_eQ</i> = 17.0 µM.</p

Fits to kinetic data from [15] on the forward operation of liver enzyme.

<p>Measured flux in arbitrary units was obtained from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001825#pone-0001825-g001" target="_blank">Figures 1</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001825#pone-0001825-g002" target="_blank">2</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001825#pone-0001825-g005" target="_blank">5</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001825#pone-0001825-g006" target="_blank">6</a> of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001825#pone.0001825-Shepherd1" target="_blank">[15]</a>. For all cases the product (CIT and COASH) concentrations are zero and total substrate and inhibitor concentrations are indicated in the figure. A and B report data obtained with no inhibitors present. C. The relative activity (normalized to its maximum) of the enzyme is plotted as functions of [ATP], [ADP], and [AMP] measured at [ACCOA] = 11 µM and [OAA] = 1.9 µM. D. The measured flux is plotted as a function of [ACCOA] at [OAA] = 34 µM with ATP, ADP, and AMP present as indicated in the figure. All data were obtained at pH = 7.4 at 25°C. Model fits are plotted as solid lines.</p