Normal Human Heart Tissue Research Articles

A method for cryopreservation and single nucleus RNA-sequencing of normal adult human interventricular septum heart tissue reveals cellular diversity and function

BackgroundSingle cell sequencing of human heart tissue is technically challenging and methods to cryopreserve heart tissue for obtaining single cell information have not been standardized. Studies published to date have used varying methods to preserve and process human heart tissue, and have generated interesting datasets, but development of a biobanking standard has not yet been achieved. Heart transcription patterns are known to be regionally diverse, and there are few single cell datasets for normal human heart tissue.MethodsUsing pig tissue, we developed a rigorous and reproducible method for tissue mincing and cryopreservation that allowed recovery of high quality single nuclei RNA. We subsequently tested this protocol on normal human heart tissue obtained from organ donors and were able to recover high quality nuclei for generation of single nuclei RNA-seq datasets, using a commercially available platform from 10× Genomics. We analyzed these datasets using standard software packages such as CellRanger and Seurat.ResultsHuman heart tissue preserved with our method consistently yielded nuclear RNA with RNA Integrity Numbers of greater than 8.5. We demonstrate the utility of this method for single nuclei RNA-sequencing of the normal human interventricular septum and delineating its cellular diversity. The human IVS showed unexpected diversity with detection of 23 distinct cell clusters that were subsequently categorized into different cell types. Cardiomyocytes and fibroblasts were the most commonly identified cell types and could be further subdivided into 5 different cardiomyocyte subtypes and 6 different fibroblast subtypes that differed by gene expression patterns. Ingenuity Pathway analysis of these gene expression patterns suggested functional diversity in these cell subtypes.ConclusionsHere we report a simple technical method for cryopreservation and subsequent nuclear isolation of human interventricular septum tissue that can be done with common laboratory equipment. We show how this method can be used to generate single nuclei transcriptomic datasets that rival those already published by larger groups in terms of cell diversity and complexity and suggest that this simple method can provide guidance for biobanking of human myocardial tissue for complex genomic analysis.

Abstract 352: The Effect of the Extracellular Matrix of Failing Hearts on Cardiac Stem Cells

Objective: Stem cell therapy to repair cardiac damage, caused by the pathological remodeling that occurs in cardiovascular disease, represents a cutting edge therapeutic option toward preventing and treating heart failure. There is a paucity of information on how the phenotypes of stem cells are altered in the “hostile” environment of pathologically remodeled hearts. The objective of this study was to (a) determine the phenotypes of cardiac stem cells cultured on failing versus normal human heart tissue, and (b) to characterize pathology of failing human heart samples. Methods: We isolated the extracellular matrix (ECM) from end-stage failing human hearts (compared to control human hearts) and used the isolated ECM to coat tissue culture plates. The ECM represents the scaffold of the heart in which stem cells will integrate, proliferate, and differentiate. We then cultured induced pluripotent stem cell-derived cardiac progenitor cells on the cardiac-ECM plates and analyzed their phenotypes (morphology, differentiation into cardiomyocytes, and gene expression). We also performed analysis of the failing cardiac tissue itself to determine the extent of fibrosis and expression of various ECM and matricellular proteins. Results: Cells cultured on failing heart ECM showed important differences compared to those on control heart ECM, including reduced cell-matrix adhesion and altered responses to beta-adrenergic stimulation. The failing heart shows increased fibrosis and differential expression of specific ECM and matricellular proteins. Significance: We will use these data to determine how best to direct cardiac stem cells used for tissue generation in severely damaged hearts toward differentiation into functioning cardiomyocytes.

Autoimmunoreactive IgGs against cardiac lipid raft-associated proteins in patients with postural orthostatic tachycardia syndrome

Lipid rafts are specialized plasma membrane microdomains that serve as platforms for integrating cellular signal transductions. We have recently reported that autoantibodies against cardiac membrane proteins are present in patients with postural orthostatic tachycardia syndrome (POTS). In this study, we examined the presence of autoimmunoreactive IgGs against lipid raft proteins in these patients. IgGs were purified from the sera of 10 patients and 7 normal controls. Cardiac lipid raft preparations were isolated from normal human heart tissue. The lipid raft-associated proteins were resolved by 2-dimensional gel electrophoresis and immunoblotted against IgGs from each subject. Protein spots that reacted specifically with patient IgGs were identified by nano-liquid chromatography-mass spectrometry/mass spectrometry. Thirty-four such protein spots, and 72 unique proteins were identified. The targets of autoimmunoreactive IgGs include proteins associated with caveolae structure, adrenergic signaling, calcium signaling, cytostructures, chaperone and energy metabolism. Multiple pathways were involved including those that regulate caveolae-mediated signaling, oxidative phosphorylation, fatty acid metabolism, protein ubiquitination, and cardiac β-adrenergic signaling. Our results suggest that cardiac lipid raft-associated proteins are targets of autoimmunoreactive IgGs from patients with POTS. Autoimmunity may play a role in the pathogenesis of POTS.

Oxidative Post-translational Modification of Tryptophan Residues in Cardiac Mitochondrial Proteins

We examined the distribution of N-formylkynurenine, a product of the dioxidation of tryptophan residues in proteins, throughout the human heart mitochondrial proteome. This oxidized amino acid is associated with a distinct subset of proteins, including an over-representation of complex I subunits as well as complex V subunits and enzymes involved in redox metabolism. No relationship was observed between the tryptophan modification and methionine oxidation, a known artifact of sample handling. As the mitochondria were isolated from normal human heart tissue and not subject to any artificially induced oxidative stress, we suggest that the susceptible tryptophan residues in this group of proteins are "hot spots" for oxidation in close proximity to a source of reactive oxygen species in respiring mitochondria.

Characterization of the human heart mitochondrial proteome.

To gain a better understanding of the critical role of mitochondria in cell function, we have compiled an extensive catalogue of the mitochondrial proteome using highly purified mitochondria from normal human heart tissue. Sucrose gradient centrifugation was employed to partially resolve protein complexes whose individual protein components were separated by one-dimensional PAGE. Total in-gel processing and subsequent detection by mass spectrometry and rigorous bioinformatic analysis yielded a total of 615 distinct protein identifications. All protein pI values, molecular weight ranges, and hydrophobicities were represented. The coverage of the known subunits of the oxidative phosphorylation machinery within the inner mitochondrial membrane was >90%. A significant proportion of identified proteins are involved in signaling, RNA, DNA, and protein synthesis, ion transport, and lipid metabolism. The biochemical roles of 19% of the identified proteins have not been defined. This database of proteins provides a comprehensive resource for the discovery of novel mitochondrial functions and pathways.

Highlights in cardiovascular effects of histamine and H1-receptor antagonists.

Despite numerous studies, the cardiac actions of histamine are still obscure. Yet, histamine could probably be clinically relevant. It is stored in large amounts in human cardiac tissue, where it is contained in the cytoplasmatic granules of mast cells. Mast cells are present in normal human heart tissue; they are more abundant in diseased human heart tissue where they lie in close proximity to blood vessels and between myocytes. The histamine content of human heart mast cells is comparable to the histamine content of lung parenchymal and skin mast cells. Ultrastructural studies confirmed the presence of mast cells around vessels and between myocytes. Consequently, these cells are easily accessible to circulating antigens, drugs and stimuli that activate the cells to release vasoactive mediators which in turn can exert significant cardiovascular effects. Histamine possesses arrhythmogenic effects and once locally released, may enhance automaticity and induce triggering activity resulting in severe tachyarrhythmias. The major arrhythmogenic effects of histamine consist in increasing sinus rate and ventricular automaticity, and in slowing atrioventricular conduction. In addition, histamine may interfere with depolarization and repolarization through its effects on calcium and potassium currents. These effects are mediated by H2-receptor. Therefore direct activation of histamine receptor can induce cardiac arrhythmias. Consequently, the interference of these histaminergic effects may explain, at least in part, the arrhythmogenic effects described for some second-generation antihistamines, such as terfenadine and astemizole. In this brief review we will discuss the cardiac effects of histamine in experimental animal models and in man, and will review data on the safety of the new second-generation antihistamines, focusing on their cardiotoxic effects.

Human Antibodies against <i>Trypanosoma cruzi</i> Exoantigens Recognizing Parasite Surface Antigens and Heart Tissue Components

This work describes the occurrence of antibodies against exoantigens of Trypanosoma cruzi in a high percentage of patients with chronic Chagas' disease. When analyzing selected groups of patients, it was observed that sera from individuals with electrocardiographic alterations showed a greater number of precipitin system and higher antibody titers than sera from patients with positive serology only. Partial characterization of exoantigens of T. cruzi was performed by isoelectric focusing and immunoblotting. Two distinct groups of antigens at pI around 5 and 6, respectively, were identified by these methods. It was also shown in absorption experiments that exoantigens of lower pI share epitopes with components of the cellular surface of epimastigotes of T. cruzi, whereas exoantigens of higher pI share epitopes with normal human heart tissue.

Class 1 MHC antigen induction on myocytes in cardiac allograft rejection

The expression of major histocompatibility complex (MHC) antigens was studied in normal human heart tissue and in endomyocardial biopsies taken after heart transplantation. Staining was performed on cryostat sections by an indirect immunoperoxidase method using monoclonal antibodies against monomorphic determinants of MHC class 1 or class 2 antigens. In the normal heart the myocytes were negative for class 1 and class 2 antigens, but all other cells expressed both types of MHC antigens. In 50 endomyocardial biopsies obtained from 16 patients after transplantation the following alterations were found: (a) de novo expression of class 1 antigen by myocytes takes place after OHT; (b) this is related to the severity and time of rejection episodes; (c) it disappears after effective steroid treatment and persists with steroid-resistant rejection; (d) an overall increase in class 2 antigen expression occurs in the graft after transplantation, not only with rejection. It is likely that the induction of the transplantation antigens on the target cells can increase their susceptibility to the immune assault. This could also be of importance in other types of inflammatory heart disease.

In Vitro Activation of Complement by Isolated Human Heart Subcellular Membranes

Abstract Activation of human complement (C) occurred in vitro when mitochondrial membranes isolated from normal human heart tissue were incubated with normal human serum. This activation, as measured by C3 depletion, was not completely inhibited by blocking classical pathway activity in serum treated with EGTA, in C2-deficient serum, or in C1-depleted serum, nor in serum heated at 50°C for 30 min to block the alternative pathway, but it could be prevented by blocking the classical and the alternative pathway simultaneously with EDTA, or by treating heated serum (50°C, 30 min) with EGTA. Factor B was converted in normal serum as well as in EGTA-treated serum, but not in EDTA-treated serum. Mitochondrial membranes had no direct enzymatic or other activity that could inactivate functionally or highly purified C4 or C3, but the membranes could bind and activate C1 either in serum or in functionally pure C1 preparations. C4 also bound to the mitochondrial membranes only in the presence of C1. These data suggest that the activation of C by heart subcellular membranes involved both the classical and the alternative pathways, that the mitochondrial membrane preparations were capable of forming stable complexes with C1 and C4, but not C3, and that the mitochondrial membrane preparations did not contain enzymes or have inherent properties that could directly cause C3 conversion.

Trace elements in normal human heart tissue

Acta Medica ScandinavicaVolume 178, Issue S439 p. 16-17 Trace elements in normal human heart tissue First published: January/December 1965 https://doi.org/10.1111/j.0954-6820.1965.tb16694.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume178, IssueS439January/December 1965Pages 16-17 RelatedInformation

IMMUNOLOGIC RELATION OF STREPTOCOCCAL AND TISSUE ANTIGENS. III. PRESENCE IN HUMAN SERA OF STREPTOCOCCAL ANTIBODY CROSS-REACTIVE WITH HEART TISSUE. ASSOCIATION WITH STREPTOCOCCAL INFECTION, RHEUMATIC FEVER, AND GLOMERULONEPHRITIS.

Sera from patients with recent streptococcal infection or non-suppurative sequelae exhibit with variable frequency a precipitin reaction in agar gel with a partially purified streptococcal antigen which has been shown previously to be immunologically related to human heart tissue. This precipitin could be absorbed from sera with normal human heart tissue homogenates but not with homogenates of other organs. Demonstration of this cross-reaction by heart absorption was found dependent both upon the serologic properties of individual sera and the nature or state of purification of the streptococcal product employed as test antigen. Evidence was obtained of a close association of heart-related and non-heart-related antigenic determinants in partially purified preparations of the streptococcal antigen by both gel diffusion and immunoelectrophoresis. On immunoelectrophoretic analysis, cross-reactive antigen exhibited a more rapid mobility toward the anode than M protein. It was destroyed by digestion with trypsin, pepsin, and chymotrypsin. Based on specific absorption tests with a Type 5 and Type 19 strain, the antigen was localized to cell walls and to a lesser extent to cell membranes of these strains. Precipitating activity related to cross-reactive antibody was localized to the immunoglobulin zone in immunoelectrophoresis. Reactive sera showed diminution or loss of serological activity following heat inactivation at 56 degrees C or after prolonged storage at 4 degrees C. Sera containing cross-reactive precipitating antibody exhibited an immunofluorescent reaction with sarcolemma of cardiac myofibers, which was inhibited by streptococcal cross-reactive antigen. By this inhibition test, the immunofluorescent reaction related to cross-reactive antibody could be distinguished from that due to other heart-reactive factors. Antibody to streptococcal cross-reactive antigen defined by precipitation-absorption tests was observed in 24 per cent of patients with recent history of uncomplicated streptococcal infection and in the majority of patients with acute rheumatic fever, rheumatic heart disease, or acute glomerulonephritis. It was observed rarely in patients with non-streptococcal related disease. These data provide evidence that induction of cross-reactive autoantibody to heart in certain individuals is associated with streptococcal infection.