14 research outputs found
Pathways to clinical CLARITY: volumetric analysis of irregular, soft, and heterogeneous tissues in development and disease
AbstractThree-dimensional tissue-structural relationships are not well captured by typical thin-section histology, posing challenges for the study of tissue physiology and pathology. Moreover, while recent progress has been made with intact methods for clearing, labeling, and imaging whole organs such as the mature brain, these approaches are generally unsuitable for soft, irregular, and heterogeneous tissues that account for the vast majority of clinical samples and biopsies. Here we develop a biphasic hydrogel methodology, which along with automated analysis, provides for high-throughput quantitative volumetric interrogation of spatially-irregular and friable tissue structures. We validate and apply this approach in the examination of a variety of developing and diseased tissues, with specific focus on the dynamics of normal and pathological pancreatic innervation and development, including in clinical samples. Quantitative advantages of the intact-tissue approach were demonstrated compared to conventional thin-section histology, pointing to broad applications in both research and clinical settings.</jats:p
Mitochondrial structure and function are not different between nonfailing donor and endâstage 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)â or FA (palmitoylcarnitine)âderived 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 0.64âfold reduced transcript levels for the mitochondrialâLD 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 mitochondrialâLD tethering, and not from intrinsic mitochondrial dysfunction.âHolzem, 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 endâstage failing human hearts. FASEB J. 30, 2698â2707 (2016). www.fasebj.orgPeer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154292/1/fsb2fj201500118r.pd
Reduced response to IKr blockade and altered hERG1a/1b stoichiometryin human heart failure
Heart failure (HF) claims 250,000 lives per year in the US, and nearly half of these deaths are sudden and presumably
due to ventricular tachyarrhythmias. QT interval and action potential (AP) prolongation are hallmark proarrhythmic
changes in the failing myocardium, which potentially result from alterations in repolarizing potassium currents.
Thus,we aimed to examinewhether decreased expression of the rapid delayed rectifier potassiumcurrent, IKr, contributes
to repolarization abnormalities in human HF. Tomap functional IKr expression across the left ventricle (LV),
we optically imaged coronary-perfused LV free wall from donor and end-stage failing human hearts. The LV wedge
preparation was used to examine transmural AP durations at 80% repolarization (APD80), and treatment with the
IKr-blocking drug, E-4031, was utilized to interrogate functional expression. We assessed the percent change in
APD80 post-IKr blockade relative to baseline APD80 (ΔAPD80) and found that ΔAPD80s are reduced in failing versus
donor hearts in each transmural region, with 0.35-, 0.43-, and 0.41-fold reductions in endo-, mid-, and epicardium,
respectively (p = 0.008, 0.037, and 0.022). We then assessed hERG1 isoform gene and protein expression levels
using qPCR and Western blot. While we did not observe differences in hERG1a or hERG1b gene expression between
donor and failing hearts, we found a shift in the hERG1a:hERG1b isoform stoichiometry at the protein level. Computer
simulations were then conducted to assess IKr block under E-4031 influence in failing and nonfailing conditions.
Our results confirmed the experimental observations and E-4031-induced relative APD80 prolongationwas greater
in normal conditions than in failing conditions, provided that the cellularmodel of HF included a significant downregulation
of IKr. In humanHF, the response to IKr blockade is reduced, suggesting decreased functional IKr expression.
This attenuated functional response is associated with altered hERG1a:hERG1b protein stoichiometry in the
failing human LV, and failing cardiomyoctye simulations support the experimental findings. Thus, of IKr protein
and functional expression may be important determinants of repolarization remodeling in the failing human LV.We thank the Translational Cardiovascular Biobank & Repository (TCBR) at Washington University for provision of donor/patient records. The TCBR is supported by the NIH/CTSA (UL1 TR000448), Children's Discovery Institute, and Richard J. Wilkinson Trust. We also thank the laboratory of Dr. Sakiyama-Elbert for the use of the StepOnePlus equipment We appreciate the critical feedback on the manuscript by Dr. Jeanne Nerbonne. This work has been supported by the National Heart, Lung & Blood Institute (NHLBI, R01 HL114395). K. Holzem has been supported by the American Heart Association (12PRE12050315) and the NHLBI (F30 HL114310).Holzem, KM.; GĂłmez GarcĂa, JF.; Glukhov, AV.; Madden, EJ.; Koppel, AC.; Ewald, GA.; TrĂŠnor Gomis, BA.... (2016). Reduced response to IKr blockade and altered hERG1a/1b stoichiometryin human heart failure. Journal of Molecular and Cellular Cardiology. 96:82-92. https://doi.org/10.1016/j.yjmcc.2015.06.008S82929
Sometimes There is MeaningâŚ
Dr. Katherine Holzem is faculty in the Department of Surgery. She shared a personal story about working through hardships, and that it\u27s okay to not be okay. (Note: content includes mention of suicide)
Excerpt:
âApril 2, 2011, coincidentally my brother Kyleâs 18th birthday, I married my husband at the MoBot. It was perfect, and in classic XXXX-family style, we found ourselves at Pin-up Bowl until 3am, with my dad fiercely defending my right to take one last turn at closing time in my wedding dress. Just days later, I broke out in this terrible rash. This didnât just happen in a vacuum. My GI system had been weird for years, but it had gotten noticeably worse leading up to the wedding. I got scoped, and my GI system was littered with immune cells.
Adverse Remodeling of the Electrophysiological Response to IschemiaâReperfusion in Human Heart Failure Is Associated With Remodeling of Metabolic Gene Expression
Right ventricular arrhythmogenesis in failing human heart: the role of conduction and repolarization remodeling
Mitochondrial structure and function are not different between nonfailing donor and endâstage 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.âHolzem, 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