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Pubmed Article
The AP-1 transcription factor c-Jun prevents stress-imposed maladaptive remodeling of the heart.
PLoS ONE
PUBLISHED: 01-01-2013
Systemic hypertension increases cardiac workload and subsequently induces signaling networks in heart that underlie myocyte growth (hypertrophic response) through expansion of sarcomeres with the aim to increase contractility. However, conditions of increased workload can induce both adaptive and maladaptive growth of heart muscle. Previous studies implicate two members of the AP-1 transcription factor family, junD and fra-1, in regulation of heart growth during hypertrophic response. In this study, we investigate the function of the AP-1 transcription factors, c-jun and c-fos, in heart growth. Using pressure overload-induced cardiac hypertrophy in mice and targeted deletion of Jun or Fos in cardiomyocytes, we show that c-jun is required for adaptive cardiac hypertrophy, while c-fos is dispensable in this context. c-jun promotes expression of sarcomere proteins and suppresses expression of extracellular matrix proteins. Capacity of cardiac muscle to contract depends on organization of principal thick and thin filaments, myosin and actin, within the sarcomere. In line with decreased expression of sarcomere-associated proteins, Jun-deficient cardiomyocytes present disarrangement of filaments in sarcomeres and actin cytoskeleton disorganization. Moreover, Jun-deficient hearts subjected to pressure overload display pronounced fibrosis and increased myocyte apoptosis finally resulting in dilated cardiomyopathy. In conclusion, c-jun but not c-fos is required to induce a transcriptional program aimed at adapting heart growth upon increased workload.
Authors: Gretchen M. Roth, David M. Bader, Elise R. Pfaltzgraff.
Published: 09-07-2014
ABSTRACT
Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.
23 Related JoVE Articles!
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Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue
Authors: Olaf Bergmann, Stefan Jovinge.
Institutions: University of Lund, University of Lund.
Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events in cardiac myocytes such as proliferation and apoptosis require an accurate identification of cardiac myocyte nuclei to analyze cellular renewal in homeostasis and in pathological conditions2. Here, we provide a method to isolate cardiomyocyte nuclei from post mortem tissue by density sedimentation and immunolabeling with antibodies against pericentriolar material 1 (PCM-1) and subsequent flow cytometry sorting. This strategy allows a high throughput analysis and isolation with the advantage of working equally well on fresh tissue and frozen archival material. This makes it possible to study material already collected in biobanks. This technique is applicable and tested in a wide range of species and suitable for multiple downstream applications such as carbon-14 dating3, cell-cycle analysis4, visualization of thymidine analogues (e.g. BrdU and IdU)4, transcriptome and epigenetic analysis.
Medicine, Issue 65, Stem Cell Biology, Cardiology, Physiology, Tissue Engineering, cardiomyocyte, post mortem, nuclei isolation, flow cytometry, pericentriolar material 1, PCM-1
4205
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Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy
Authors: Stephen C. Kolwicz Jr., Rong Tian.
Institutions: University of Washington School of Medicine.
Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of cardiovascular disease. Although molecular biology is necessary in identifying key changes in the signaling pathway, it is not a surrogate for functional significance. While physiology can provide answers to the question of function, combining physiology with biochemical assessment of metabolites in the intact, beating heart allows for a complete picture of cardiac function and energetics. For years, our laboratory has utilized isolated heart perfusions combined with nuclear magnetic resonance (NMR) spectroscopy to accomplish this task. Left ventricular function is assessed by Langendorff-mode isolated heart perfusions while cardiac energetics is measured by performing 31P magnetic resonance spectroscopy of the perfused hearts. With these techniques, indices of cardiac function in combination with levels of phosphocreatine and ATP can be measured simultaneously in beating hearts. Furthermore, these parameters can be monitored while physiologic or pathologic stressors are instituted. For example, ischemia/reperfusion or high workload challenge protocols can be adopted. The use of aortic banding or other models of cardiac pathology are apt as well. Regardless of the variants within the protocol, the functional and energetic significance of molecular modifications of transgenic mouse models can be adequately described, leading to new insights into the associated enzymatic and metabolic pathways. Therefore, 31P NMR spectroscopy in the isolated perfused heart is a valuable research technique in animal models of cardiovascular disease.
Medicine, Issue 42, cardiac physiology, high energy phosphate, phosphocreatine, ATP
2069
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Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes
Authors: Lars G. Eckerle, Stephan B. Felix, Lars R. Herda.
Institutions: University Medicine Greifswald.
Dilated cardiomyopathy (DCM) is one of the main causes for heart failure in younger adults1. Although genetic disposition and exposition to toxic substances are known causes for this disease in about one third of the patients, the origin of DCM remains largely unclear. In a substantial number of these patients, autoantibodies against cardiac epitopes have been detected and are suspected to play a pivotal role in the onset and progression of the disease2,3. The importance of cardiac autoantibodies is underlined by a hemodynamic improvement observed in DCM patients after elimination of autoantibodies by immunoadsorption3-5. A variety of specific antigens have already been identified2,3 and antibodies against these targets may be detected by immunoassays. However, these assays cannot discriminate between stimulating (and therefore functionally effective) and blocking autoantibodies. There is increasing evidence that this distinction is crucial6,7. It can also be assumed that the targets for a number of cardiotropic antibodies are still unidentified and therefore cannot be detected by immunoassays. Therefore, we established a method for the detection of functionally active cardiotropic antibodies, independent of their respective antigen. The background for the method is the high homology usually observed for functional regions of cardiac proteins in between mammals8,9. This suggests that cardiac antibodies directed against human antigens will cross-react with non-human target cells, which allows testing of IgG from DCM patients on adult rat cardiomyocytes. Our method consists of 3 steps: first, IgG is isolated from patient plasma using sepharose coupled anti-IgG antibodies obtained from immunoadsorption columns (PlasmaSelect, Teterow, Germany). Second, adult cardiomyocytes are isolated by collagenase perfusion in a Langendorff perfusion apparatus using a protocol modified from previous works10,11. The obtained cardiomyocytes are attached to laminin-coated chambered coverglasses and stained with Fura-2, a calcium-selective fluorescent dye which can be easily brought into the cell to observe intracellular calcium (Ca2+) contents12. In the last step, the effect of patient IgG on the cell shortening and Ca2+ transients of field stimulated cardiomyocytes is monitored online using a commercial myocyte calcium and contractility monitoring system (IonOptix, Milton, MA, USA) connected to a standard inverse fluorescent microscope.
Immunology, Issue 73, Medicine, Cellular Biology, Molecular Biology, Biomedical Engineering, Physiology, Anatomy, Cardiology, cardiomyocytes, cell shortening, intracellular Ca2+, Fura-2, antibodies, dilated cardiomyopathy, DCM, IgG, cardiac proteins, Langendorff perfusion, electrode, immunoassay, assay, cell culture, animal model
4237
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Fluorescent Labeling of Drosophila Heart Structures
Authors: Nakissa N. Alayari, Georg Vogler, Ouarda Taghli-Lamallem, Karen Ocorr, Rolf Bodmer, Anthony Cammarato.
Institutions: San Diego State University, The Sanford Burnham Institute for Medical Research.
The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of α-actinin in larval hearts. Additionally, we perform labeling of F-actin and α-actinin in myosin-GFP expressing adult flies and of α-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.
Cellular Biology, Issue 32, Cardiac, cardiomyopathy, dorsal vessel, fluorescence, staining, GFP, larva, immunohistochemistry, microscopy, imaging
1423
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Reduction in Left Ventricular Wall Stress and Improvement in Function in Failing Hearts using Algisyl-LVR
Authors: Lik Chuan Lee, Zhang Zhihong, Andrew Hinson, Julius M. Guccione.
Institutions: UCSF/VA Medical Center, LoneStar Heart, Inc..
Injection of Algisyl-LVR, a treatment under clinical development, is intended to treat patients with dilated cardiomyopathy. This treatment was recently used for the first time in patients who had symptomatic heart failure. In all patients, cardiac function of the left ventricle (LV) improved significantly, as manifested by consistent reduction of the LV volume and wall stress. Here we describe this novel treatment procedure and the methods used to quantify its effects on LV wall stress and function. Algisyl-LVR is a biopolymer gel consisting of Na+-Alginate and Ca2+-Alginate. The treatment procedure was carried out by mixing these two components and then combining them into one syringe for intramyocardial injections. This mixture was injected at 10 to 19 locations mid-way between the base and apex of the LV free wall in patients. Magnetic resonance imaging (MRI), together with mathematical modeling, was used to quantify the effects of this treatment in patients before treatment and at various time points during recovery. The epicardial and endocardial surfaces were first digitized from the MR images to reconstruct the LV geometry at end-systole and at end-diastole. Left ventricular cavity volumes were then measured from these reconstructed surfaces. Mathematical models of the LV were created from these MRI-reconstructed surfaces to calculate regional myofiber stress. Each LV model was constructed so that 1) it deforms according to a previously validated stress-strain relationship of the myocardium, and 2) the predicted LV cavity volume from these models matches the corresponding MRI-measured volume at end-diastole and end-systole. Diastolic filling was simulated by loading the LV endocardial surface with a prescribed end-diastolic pressure. Systolic contraction was simulated by concurrently loading the endocardial surface with a prescribed end-systolic pressure and adding active contraction in the myofiber direction. Regional myofiber stress at end-diastole and end-systole was computed from the deformed LV based on the stress-strain relationship.
Medicine, Issue 74, Biomedical Engineering, Anatomy, Physiology, Biophysics, Molecular Biology, Surgery, Cardiology, Cardiovascular Diseases, bioinjection, ventricular wall stress, mathematical model, heart failure, cardiac function, myocardium, left ventricle, LV, MRI, imaging, clinical techniques
50096
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Myocardial Infarction and Functional Outcome Assessment in Pigs
Authors: Stefan Koudstaal, Sanne J. Jansen of Lorkeers, Johannes M.I.H. Gho, Gerardus P.J van Hout, Marlijn S. Jansen, Paul F. Gründeman, Gerard Pasterkamp, Pieter A. Doevendans, Imo E. Hoefer, Steven A.J. Chamuleau.
Institutions: University Medical Center Utrecht, Interuniversity Cardiology Institute of the Netherlands.
Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.
Medicine, Issue 86, myocardial infarction (MI), AMI, large animal model, pig, translational medicine, ischemic heart disease
51269
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Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice
Authors: Wen-Chi Chen, Sung-Hyun Park, Carol Hoffman, Cecil Philip, Linda Robinson, James West, Gabriele Grunig.
Institutions: New York University School of Medicine, Tuxedo, Vanderbilt University Medical Center, New York University School of Medicine.
The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is a common modifier of heart and lung function, by elaborating cellular infiltration, production of cytokines and growth factors, and by initiating remodeling processes 1. Compared to the left ventricle, the right ventricle is a low-pressure pump that operates in a relatively narrow zone of pressure changes. Increased pulmonary artery pressures are associated with increased pressure in the lung vascular bed and pulmonary hypertension 2. Pulmonary hypertension is often associated with inflammatory lung diseases, for example chronic obstructive pulmonary disease, or autoimmune diseases 3. Because pulmonary hypertension confers a bad prognosis for quality of life and life expectancy, much research is directed towards understanding the mechanisms that might be targets for pharmaceutical intervention 4. The main challenge for the development of effective management tools for pulmonary hypertension remains the complexity of the simultaneous understanding of molecular and cellular changes in the right heart, the lungs and the immune system. Here, we present a procedural workflow for the rapid and precise measurement of pressure changes in the right heart of mice and the simultaneous harvest of samples from heart, lungs and immune tissues. The method is based on the direct catheterization of the right ventricle via the jugular vein in close-chested mice, first developed in the late 1990s as surrogate measure of pressures in the pulmonary artery5-13. The organized team-approach facilitates a very rapid right heart catheterization technique. This makes it possible to perform the measurements in mice that spontaneously breathe room air. The organization of the work-flow in distinct work-areas reduces time delay and opens the possibility to simultaneously perform physiology experiments and harvest immune, heart and lung tissues. The procedural workflow outlined here can be adapted for a wide variety of laboratory settings and study designs, from small, targeted experiments, to large drug screening assays. The simultaneous acquisition of cardiac physiology data that can be expanded to include echocardiography5,14-17 and harvest of heart, lung and immune tissues reduces the number of animals needed to obtain data that move the scientific knowledge basis forward. The procedural workflow presented here also provides an ideal basis for gaining knowledge of the networks that link immune, lung and heart function. The same principles outlined here can be adapted to study other or additional organs as needed.
Immunology, Issue 71, Medicine, Anatomy, Physiology, Cardiology, Surgery, Cardiovascular Abnormalities, Inflammation, Respiration Disorders, Immune System Diseases, Cardiac physiology, mouse, pulmonary hypertension, right heart function, lung immune response, lung inflammation, lung remodeling, catheterization, mice, tissue, animal model
50023
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Evaluation of a Novel Laser-assisted Coronary Anastomotic Connector - the Trinity Clip - in a Porcine Off-pump Bypass Model
Authors: David Stecher, Glenn Bronkers, Jappe O.T. Noest, Cornelis A.F. Tulleken, Imo E. Hoefer, Lex A. van Herwerden, Gerard Pasterkamp, Marc P. Buijsrogge.
Institutions: University Medical Center Utrecht, Vascular Connect b.v., University Medical Center Utrecht, University Medical Center Utrecht.
To simplify and facilitate beating heart (i.e., off-pump), minimally invasive coronary artery bypass surgery, a new coronary anastomotic connector, the Trinity Clip, is developed based on the excimer laser-assisted nonocclusive anastomosis technique. The Trinity Clip connector enables simplified, sutureless, and nonocclusive connection of the graft to the coronary artery, and an excimer laser catheter laser-punches the opening of the anastomosis. Consequently, owing to the complete nonocclusive anastomosis construction, coronary conditioning (i.e., occluding or shunting) is not necessary, in contrast to the conventional anastomotic technique, hence simplifying the off-pump bypass procedure. Prior to clinical application in coronary artery bypass grafting, the safety and quality of this novel connector will be evaluated in a long-term experimental porcine off-pump coronary artery bypass (OPCAB) study. In this paper, we describe how to evaluate the coronary anastomosis in the porcine OPCAB model using various techniques to assess its quality. Representative results are summarized and visually demonstrated.
Medicine, Issue 93, Anastomosis, coronary, anastomotic connector, anastomotic coupler, excimer laser-assisted nonocclusive anastomosis (ELANA), coronary artery bypass graft (CABG), off-pump coronary artery bypass (OPCAB), beating heart surgery, excimer laser, porcine model, experimental, medical device
52127
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Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos
Authors: Qiaozhi Wei, Nancy R. Manley, Brian G. Condie.
Institutions: University of Georgia.
Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.
Developmental Biology, Issue 56, transcriptome, in situ hybridization, mouse embryo, gene expression, transcripts, mRNA, in vitro transcription, riboprobe
2797
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Compensatory Limb Use and Behavioral Assessment of Motor Skill Learning Following Sensorimotor Cortex Injury in a Mouse Model of Ischemic Stroke
Authors: Abigail L. Kerr, Kelly A. Tennant.
Institutions: Illinois Wesleyan University, University of Victoria.
Mouse models have become increasingly popular in the field of behavioral neuroscience, and specifically in studies of experimental stroke. As models advance, it is important to develop sensitive behavioral measures specific to the mouse. The present protocol describes a skilled motor task for use in mouse models of stroke. The Pasta Matrix Reaching Task functions as a versatile and sensitive behavioral assay that permits experimenters to collect accurate outcome data and manipulate limb use to mimic human clinical phenomena including compensatory strategies (i.e., learned non-use) and focused rehabilitative training. When combined with neuroanatomical tools, this task also permits researchers to explore the mechanisms that support behavioral recovery of function (or lack thereof) following stroke. The task is both simple and affordable to set up and conduct, offering a variety of training and testing options for numerous research questions concerning functional outcome following injury. Though the task has been applied to mouse models of stroke, it may also be beneficial in studies of functional outcome in other upper extremity injury models.
Behavior, Issue 89, Upper extremity impairment, Murine model, Rehabilitation, Reaching, Non-paretic limb training, Good limb training, Less-affected limb training, Learned non-use, Pasta matrix reaching task
51602
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Ascending Aortic Constriction in Rats for Creation of Pressure Overload Cardiac Hypertrophy Model
Authors: Ajith Kumar GS, Binil Raj, Santhosh Kumar S, Sanjay G, Chandrasekharan Cheranellore Kartha.
Institutions: Rajiv Gandhi Centre for Biotechnology, Rajiv Gandhi Centre for Biotechnology, Sree Chitra Tirunal Institute for Medical Sciences & Technology.
Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart failure. Here, we describe a detailed surgical procedure for creating pressure overload and cardiac hypertrophy in rats by constriction of the ascending aorta using a small metallic clip. After anesthesia, the trachea is intubated by inserting a cannula through a half way incision made between two cartilage rings of trachea. Then a skin incision is made at the level of the second intercostal space on the left chest wall and muscle layers are cleared to locate the ascending portion of aorta. The ascending aorta is constricted to 50–60% of its original diameter by application of a small sized titanium clip. Following aortic constriction, the second and third ribs are approximated with prolene sutures. The tracheal cannula is removed once spontaneous breathing was re-established. The animal is allowed to recover on the heating pad by gradually lowering anesthesia. The intensity of pressure overload created by constriction of the ascending aorta is determined by recording the pressure gradient using trans-thoracic two dimensional Doppler-echocardiography. Overall this protocol is useful to study the remodeling events and contractile properties of the heart during the gradual onset and progression from compensated cardiac hypertrophy to heart failure stage.
Medicine, Issue 88, ascending aorta, cardiac hypertrophy, pressure overload, aortic constriction, thoracotomy, surgical model.
50983
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Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes
Authors: Lu Chen, Soon-Keat Ooi, Joan W. Conaway, Ronald C. Conaway.
Institutions: Stowers Institute for Medical Research, Kansas University Medical Center.
Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.
Biochemistry, Issue 92, chromatin remodeling, INO80, SNF2 family ATPase, biochemical assays, ATPase, nucleosome remodeling, nucleosome binding
51721
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Isolation and Culture of Neonatal Mouse Cardiomyocytes
Authors: Elisabeth Ehler, Thomas Moore-Morris, Stephan Lange.
Institutions: King’s College London, University of California San Diego .
Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes. The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.
Cellular Biology, Issue 79, Biomedical Engineering, Bioengineering, Molecular Biology, Cell Culture Techniques, Primary Cell Culture, Cell Culture Techniques, Primary Cell Culture, Cell Culture Techniques, Primary Cell Culture, Cell Culture Techniques, Disease Models, Animal, Models, Cardiovascular, Cell Biology, neonatal mouse, cardiomyocytes, isolation, culture, primary cells, NMC, heart cells, animal model
50154
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Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy
Authors: Daxiang Li, Jian Wu, Yan Bai, Xiaochen Zhao, Lijun Liu.
Institutions: University of Toledo College of Medicine and Life Sciences, University of Toledo College of Medicine and Life Sciences.
Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes’ terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.
Basic Protocol, Issue 87, adult mouse cardiomyocytes, collagenase, isolation, primary cell culture
51357
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High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry
Authors: Subarna Bhattacharya, Paul W. Burridge, Erin M. Kropp, Sandra L. Chuppa, Wai-Meng Kwok, Joseph C. Wu, Kenneth R. Boheler, Rebekah L. Gundry.
Institutions: Medical College of Wisconsin, Stanford University School of Medicine, Medical College of Wisconsin, Hong Kong University, Johns Hopkins University School of Medicine, Medical College of Wisconsin.
There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle “in a dish” for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.
Cellular Biology, Issue 91, human induced pluripotent stem cell, flow cytometry, directed differentiation, cardiomyocyte, IRX4, TNNI3, TNNT2, MCL2v, MLC2a
52010
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Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles
Authors: Eva Wagner, Sören Brandenburg, Tobias Kohl, Stephan E. Lehnart.
Institutions: Heart Research Center Goettingen, University Medical Center Goettingen, German Center for Cardiovascular Research (DZHK) partner site Goettingen, University of Maryland School of Medicine.
In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca2+ release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.
Bioengineering, Issue 92, cardiac myocyte, atria, ventricle, heart, primary cell isolation, fluorescence microscopy, membrane tubule, transverse-axial tubule system, image analysis, image processing, T-tubule, collagenase
51823
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Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples
Authors: Raffaele Coppini, Cecila Ferrantini, Alessandro Aiazzi, Luca Mazzoni, Laura Sartiani, Alessandro Mugelli, Corrado Poggesi, Elisabetta Cerbai.
Institutions: University of Florence, University of Florence.
Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models. Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method. The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.
Medicine, Issue 86, cardiology, cardiac cells, electrophysiology, excitation-contraction coupling, action potential, calcium, myocardium, hypertrophic cardiomyopathy, cardiac patients, cardiac disease
51116
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Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes
Authors: Evan Lee Graham, Cristina Balla, Hannabeth Franchino, Yonathan Melman, Federica del Monte, Saumya Das.
Institutions: Beth Israel Deaconess Medical Center, Harvard Medical School, Sapienza University.
The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.
Cellular Biology, Issue 79, Medicine, Cardiology, Cellular Biology, Anatomy, Physiology, Mice, Ion Channels, Primary Cell Culture, Cardiac Electrophysiology, adult mouse cardiomyocytes, cell isolation, IonOptix, Cell Culture, adenoviral transfection, patch clamp, fluorescent nanosensor
50289
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Methods to Assess Subcellular Compartments of Muscle in C. elegans
Authors: Christopher J. Gaffney, Joseph J. Bass, Thomas F. Barratt, Nathaniel J. Szewczyk.
Institutions: University of Nottingham.
Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm’s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.
Developmental Biology, Issue 93, Physiology, C. elegans, muscle, mitochondria, sarcomeres, ageing
52043
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Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure
Authors: Ilayaraja Muthuramu, Marleen Lox, Frank Jacobs, Bart De Geest.
Institutions: Catholic University of Leuven.
Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure. Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. This architectural remodelling of the left ventricle is induced by injury (e.g., myocardial infarction), by pressure overload (e.g., systemic arterial hypertension or aortic stenosis), or by volume overload. Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure. A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion. In contrast, the infarct area at 24 hr after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling. Besides the model of permanent ligation of the left anterior descending coronary artery, the technique of invasive hemodynamic measurements in mice is presented in detail.
Medicine, Issue 94, Myocardial infarction, cardiac remodelling, infarct expansion, heart failure, cardiac function, invasive hemodynamic measurements
52206
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Quantitative Analysis of Chromatin Proteomes in Disease
Authors: Emma Monte, Haodong Chen, Maria Kolmakova, Michelle Parvatiyar, Thomas M. Vondriska, Sarah Franklin.
Institutions: David Geffen School of Medicine at UCLA, David Geffen School of Medicine at UCLA, David Geffen School of Medicine at UCLA, Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah.
In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due to the high percentage of binucleated cells,1 the predominantly heterochromatic state of the DNA, and the non-dividing nature of the cardiomyocyte which renders adult nuclei in a permanent state of interphase.2 Transcriptional regulation during development and disease have been well studied in this organ,3-5 but what remains relatively unexplored is the role played by the nuclear proteins responsible for DNA packaging and expression, and how these proteins control changes in transcriptional programs that occur during disease.6 In the developed world, heart disease is the number one cause of mortality for both men and women.7 Insight on how nuclear proteins cooperate to regulate the progression of this disease is critical for advancing the current treatment options. Mass spectrometry is the ideal tool for addressing these questions as it allows for an unbiased annotation of the nuclear proteome and relative quantification for how the abundance of these proteins changes with disease. While there have been several proteomic studies for mammalian nuclear protein complexes,8-13 until recently14 there has been only one study examining the cardiac nuclear proteome, and it considered the entire nucleus, rather than exploring the proteome at the level of nuclear sub compartments.15 In large part, this shortage of work is due to the difficulty of isolating cardiac nuclei. Cardiac nuclei occur within a rigid and dense actin-myosin apparatus to which they are connected via multiple extensions from the endoplasmic reticulum, to the extent that myocyte contraction alters their overall shape.16 Additionally, cardiomyocytes are 40% mitochondria by volume17 which necessitates enrichment of the nucleus apart from the other organelles. Here we describe a protocol for cardiac nuclear enrichment and further fractionation into biologically-relevant compartments. Furthermore, we detail methods for label-free quantitative mass spectrometric dissection of these fractions-techniques amenable to in vivo experimentation in various animal models and organ systems where metabolic labeling is not feasible.
Medicine, Issue 70, Molecular Biology, Immunology, Genetics, Genomics, Physiology, Protein, DNA, Chromatin, cardiovascular disease, proteomics, mass spectrometry
4294
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Transverse Aortic Constriction in Mice
Authors: Angela C. deAlmeida, Ralph J. van Oort, Xander H.T. Wehrens.
Institutions: Baylor College of Medicine (BCM), Baylor College of Medicine (BCM).
Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart failure.1 TAC initially leads to compensated hypertrophy of the heart, which often is associated with a temporary enhancement of cardiac contractility. Over time, however, the response to the chronic hemodynamic overload becomes maladaptive, resulting in cardiac dilatation and heart failure.2 The murine TAC model was first validated by Rockman et al.1, and has since been extensively used as a valuable tool to mimic human cardiovascular diseases and elucidate fundamental signaling processes involved in the cardiac hypertrophic response and heart failure development. When compared to other experimental models of heart failure, such as complete occlusion of the left anterior descending (LAD) coronary artery, TAC provides a more reproducible model of cardiac hypertrophy and a more gradual time course in the development of heart failure. Here, we describe a step-by-step procedure to perform surgical TAC in mice. To determine the level of pressure overload produced by the aortic ligation, a high frequency Doppler probe is used to measure the ratio between blood flow velocities in the right and left carotid arteries.3, 4 With surgical survival rates of 80-90%, transverse aortic banding is an effective technique of inducing left ventricular hypertrophy and heart failure in mice.
Medicine, Issue 38, Aorta, heart failure, hypertrophy, mouse, pressure-overload
1729
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Immunostaining of Dissected Zebrafish Embryonic Heart
Authors: Jingchun Yang, Xiaolei Xu.
Institutions: Mayo Clinic College of Medicine.
Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.
Developmental Biology, Issue 59, Zebrafish, Danio rerio, Embryonic Heart, Cardiology, Dissection, Immunostaining
3510
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