Isolation Of Cardiomyocytes Research Articles

Bottom-up proteomic analysis of human adult cardiac tissue and isolated cardiomyocytes

The heart is composed of multiple cell types, each with a specific function. Cell-type-specific approaches are necessary for defining the intricate molecular mechanisms underlying cardiac development, homeostasis, and pathology. While single-cell RNA-seq studies are beginning to define the chamber-specific cellular composition of the heart, our views of the proteome are more limited because most proteomics studies have utilized homogenized human cardiac tissue. To promote future cell-type specific analyses of the human heart, we describe the first method for cardiomyocyte isolation from cryopreserved human cardiac tissue followed by flow cytometry for purity assessment. We also describe a facile method for preparing isolated cardiomyocytes and whole cardiac tissue homogenate for bottom-up proteomic analyses. Prior experience in dissociating cardiac tissue or proteomics is not required to execute these methods. We compare different sample preparation workflows and analysis methods to demonstrate how these can impact the depth of proteome coverage achieved. We expect this how-to guide will serve as a starting point for investigators interested in general and cell-type-specific views of the cardiac proteome.

Isolation and Culture of Neonatal Murine Primary Cardiomyocytes.

The cardiomyocyte is the main cell type in the heart responsible for its contractile function. Culturing primary cardiomyocytes from mammalian sources to study their function remains challenging as they are terminally differentiated and cease to multiply soon after birth. The major technical hurdles associated with primary cardiomyocyte culture include attaining high yields, obtaining healthy/viable cells that show spontaneous contractions upon culture, and avoiding contamination by non-myocyte cardiac cell types such as fibroblasts and endothelial cells. The yield and the quality of the cardiomyocytes obtained are impacted by a variety of factors, such as the purity of the reagents, composition of the digestion mixture, the digestion conditions, and the temperature of the tissue during different steps of isolation. Here, we provide a simplified workflow to isolate, culture, and maintain neonatal primary cardiomyocytes from rats/mice in culture dishes, which can then be used to study, for instance, cardiac hypertrophy and drug-induced cardiotoxicity. © 2021 Wiley Periodicals LLC. Basic Protocol: Isolation and culture of primary cardiomyocytes from rat/mouse pups Support Protocol: Coating of tissue culture plates with extracellular matrix substrates for efficient cardiomyocyte attachment.

An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice

In basic research using mouse heart, isolating viable individual cardiomyocytes is a crucial technical step to overcome. Traditionally, isolating cardiomyocytes from rabbits, guinea pigs or rats has been performed via retrograde perfusion of the heart with enzymes using a Langendorff apparatus. However, a high degree of skill is required when this method is used with a small mouse heart. An antegrade perfusion method that does not use a Langendorff apparatus was recently reported for the isolation of mouse cardiomyocytes. We herein report a complete protocol for the improved antegrade perfusion of the excised heart to isolate individual heart cells from adult mice (8 - 108 weeks old). Antegrade perfusion is performed by injecting perfusate near the apex of the left ventricle of the excised heart, the aorta of which was clamped, using an infusion pump. All procedures are carried out on a pre-warmed heater mat under a microscope, which allows for the injection and perfusion processes to be monitored. The results suggest that ventricular and atrial myocytes, and fibroblasts can be well isolated from a single adult mouse simultaneously.

An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice.

In basic research using mouse heart, isolating viable individual cardiomyocytes is a crucial technical step to overcome. Traditionally, isolating cardiomyocytes from rabbits, guinea pigs or rats has been performed via retrograde perfusion of the heart with enzymes using a Langendorff apparatus. However, a high degree of skill is required when this method is used with a small mouse heart. An antegrade perfusion method that does not use a Langendorff apparatus was recently reported for the isolation of mouse cardiomyocytes. We herein report a complete protocol for the improved antegrade perfusion of the excised heart to isolate individual heart cells from adult mice (8 - 108 weeks old). Antegrade perfusion is performed by injecting perfusate near the apex of the left ventricle of the excised heart, the aorta of which was clamped, using an infusion pump. All procedures are carried out on a pre-warmed heater mat under a microscope, which allows for the injection and perfusion processes to be monitored. The results suggest that ventricular and atrial myocytes, and fibroblasts can be well isolated from a single adult mouse simultaneously.

Isolation and characterization of human embryonic stem cell-derived heart field-specific cardiomyocytes unravels new insights into their transcriptional and electrophysiological profiles.

We prospectively isolate and characterize first and second heart field- and nodal-like cardiomyocytes using a double reporter line from human embryonic stem cells. Our double reporter line utilizes two important transcription factors in cardiac development, TBX5 and NKX2-5. TBX5 expression marks first heart field progenitors and cardiomyocytes while NKX2-5 is expressed in nearly all myocytes of the developing heart (excluding nodal cells). We address the shortcomings of prior work in the generation of heart field-specific cardiomyocytes from induced pluripotent stem cells and provide a comprehensive early developmental transcriptomic as well as electrophysiological analyses of these three populations. Transcriptional, immunocytochemical, and functional studies support the cellular identities of isolated populations based on the expression pattern of NKX2-5 and TBX5. Importantly, bulk and single-cell RNA sequencing analyses provide evidence of unique molecular signatures of isolated first and second heart field cardiomyocytes, as well as nodal-like cells. Extensive electrophysiological analyses reveal dominant atrial action potential phenotypes in first and second heart fields in alignment with our findings in single-cell RNA sequencing. Lastly, we identify two novel surface markers, POPDC2 and CORIN, that enable purification of cardiomyocytes and first heart field cardiomyocytes, respectively. We describe a high-yield approach for isolation and characterization of human embryonic stem cell-derived heart field-specific and nodal-like cardiomyocytes. Obtaining enriched populations of these different cardiomyocyte subtypes increases the resolution of gene expression profiling during early cardiogenesis, arrhythmia modelling, and drug screening. This paves the way for the development of effective stem cell therapy to treat diseases that affect specific regions of the heart- or chamber-specific congenital heart defects.

Isolation of Adult Mouse Cardiomyocytes Using Langendorff Perfusion Apparatus.

Heart disease is one of the leading causes of death in the United States. Isolation and culture adult cardiomyocytes are important for studying cardiomyocyte contractility, heart hypertrophy, and cardiac failure. In contrast to neonatal cardiomyocyte isolation, adult mice cardiomyocytes isolation is challenging due to firm connections among cardiomyocytes through intercalated discs. The availability of newly generated genetically modified mouse lines requires to establish protocols to isolation and culture adult mouse cardiomyocyte for in vitro studies. In this manuscript, we described a straightforward method of isolating adult mouse cardiomyocytes using Langendorff perfusion apparatus. Briefly, the hearts were harvested from adult mice and the heart was mounted to Lagendorff apparatus. After perfusion with calcium depletion and collagenase digestion, the left ventricles were minced and filtered. Lastly, the separated cardiomyocytes were treated with CaCl2. The isolated cardiac myocytes can be utilized in a broad range of experiments including screening for drugs.

Isolation of Cardiomyocytes Derived from Human Pluripotent Stem Cells Using miRNA Switches.

The most common method for isolating cells of interest is an antibody method that recognizes cell surface antigens. However, specific surface antigens for many cell types have not been identified. We have developed the microRNA (miRNA)-responsive synthetic mRNA systems (miRNA switches), which isolate target cells based on endogenous miRNA activity. In this chapter, we describe protocols for isolating human pluripotent stem cell (hPSC)-derived cardiomyocytes using miRNA switches with or without cell sorting.

A combined Langendorff-injection technique for simultaneous isolation of single cardiomyocytes from atria and ventricles of the rat heart

Single cardiomyocytes are widely used for investigations of the cellular and molecular mechanisms of regulation and modulation of cardiac performance. Intact cardiomyocytes allow one to study in detail cell function avoiding the effects of extracellular matrix and neighboring cells. The most established protocols of cardiomyocyte isolation are based on the isolated heart perfusion using a Langendorff-apparatus or on intraventricular perfusion using a syringe. However, the yield of single cardiomyocytes obtained by these methods may be low due to the cell injury following non-uniform enzyme digestion of connective tissue in different heart chambers. Moreover, isolation of atrial cardiomyocytes is challenging because of their small size and complex geometric shape. Here we present a new protocol for simultaneous isolation of high quality cardiomyocytes from the atria, ventricular free walls and interventricular septum. The protocol is based on the combination of the Langendorff perfusion method with the intraventricular and intra-atrial injection technique taking into account the collagen content variation between the different heart chambers. Obtained cells demonstrate rod-shaped morphology, a clear and regular sarcomere striation pattern and rat-specific frequency-dependence of contraction and calcium transient parameters. Our protocol provides gentle cell isolation that increases the yield of single cardiomyocytes suitable for biophysical researches .

Generation of a human iPSC line GIBHi002-A-2 with a dual-reporter for NKX2-5 using TALENs.

The human transcription factor NKX2-5 plays an important role in cardiac formation and development, and thus it can be used for isolation of cardiomyocytes (CMs) differentiated from human pluripotent stem cells (hPSCs). Here, we knocked-in enhanced GFP (eGFP) and Pac (a puromycin resistant gene; PuroR) into the exon 1 coding region of NKX2-5 from a human iPSC line iPSN0003 using TALENs. The generated GIBHi002-A-2 enables us to monitor and optimize cardiac differentiation procedures via the cardiac progenitor cells (CPCs), as well as to isolate iPSC-derived CMs for drug screening.

Optimized Langendorff perfusion system for cardiomyocyte isolation in adult mouse heart.

With the rapid development of single‐cell sequencing technology, the Langendorff perfusion system has emerged as a common approach to decompose cardiac tissue and obtain living cardiomyocytes to study cardiovascular disease with the mechanism of cardiomyocyte biology. However, the traditional Langendorff perfusion system is difficult to master, and further, the viability and purity of cardiomyocytes are frequently unable to meet sequencing requirements due to complicated devices and manipulate processes. Here, we provide an optimized Langendorff perfusion system with a simplified and standardized operating protocol which utilizes gravity as the perfusion pressure, includes a novel method for bubbles removing and standardizes the criteria for termination of digestion. We obtained stable cardiomyocyte with high viability and purity after multiple natural gravity sedimentation. The combination of the optimized Langendorff perfusion system and the multiple natural gravity sedimentation provides a stable system for isolating adult mouse heart, which will provide higher‐quality cardiomyocytes for further experiments.

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes.

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression. The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

Isolation of Cardiomyocytes from Fixed Hearts for Immunocytochemistry and Ploidy Analysis.

The adult mammalian heart is composed of various cell types including cardiomyocytes, endothelial cells and fibroblasts. Since it is difficult to reliably identify nuclei of cardiomyocytes on histological sections, many groups rely on isolating viable cardiomyocytes prior to fixation to perform immunostaining. However, these live cardiomyocyte isolation techniques require optimization to maximize the yield, viability and quality of the samples, with inherent fluctuations from sample to sample despite maximum optimization. Here, we report a reproducible protocol, involving fixation prior to enzymatic digestion of the heart, which leads to maximum yield while preserving the in vivo morphology of individual cardiomyocytes. We further developed an automated analysis platform to determine the number of nuclei and DNA content per nucleus for individual cardiomyocytes. After exposing the chest cavity, the heart was arrested in diastole by perfusion with 60 mM KCl in PBS. Next, the heart was fixed in 4% paraformaldehyde (PFA) solution, and then digested with 60 mg/mL collagenase solution. After digestions, cells were singularized by trituration, and the cardiomyocyte fraction was enriched via differential centrifugation. Isolated cardiomyocytes were stained for Troponin T and α-actinin to assess purity of the obtained population. Furthermore, we developed an image analysis platform to determine cardiomyocyte nucleation and ploidy status following DAPI staining. Image based ploidy assessments led to consistent and reproducible results. Thus, with this protocol, it is possible to preserve native morphology of individual cardiomyocytes to allow immunocytochemistry and DNA content analysis while achieving maximum yield.

Isolation of Cardiomyocytes from Fixed Hearts for Immunocytochemistry and Ploidy Analysis

Isolation of Cardiomyocytes from Fixed Hearts for Immunocytochemistry and Ploidy Analysis

Impact of cardiac sympathetic denervation on IH-induced ischemic cardiomyopathy aggravation

Patients with sleep disordered breathing (SDB) exhibit poor prognosis after myocardial infarction (MI). Intermittent hypoxia (IH), the hallmark feature of SDB, enhances sympathetic activity, depletes cardiac adrenergic reserve and accelerates cardiac remodeling and dysfunction in a rat model of ischemic cardiomyopathy. In the present study, we aim at investigating whether specific cardiac sympathetic denervation (CSD) counteracts the IH-induced adrenergic reserve depletion and ischemic cardiomyopathy aggravation. MI is induced in male Wistar rats by permanent ligation of the left coronary artery and CSD by ablation of the left middle cervical and stellate ganglions. After surgery, rats are exposed to 6 and 14 weeks IH (21–5% FiO2, 60 s cycle, 8 h/day) or normoxia (N). Cardiac function and remodeling are evaluated by echography. Cardiac sympathetic activity is assessed by spectral analysis of heart rate variability on conscious rats (Etisense). Ultimately, hearts are withdrawn for cardiomyocyte isolation and calcium transient analysis, for assessment of calcium and adrenergic signalling pathways (western blot) and for histological analysis of fibrosis (Sirius red), hypertrophy (WGA) and cardiac sympathetic innervation (Tyrosine-hydroxylase staining). CSD prevents the IH-induced blunted cardiomyocyte response to isoproterenol challenge (ISO). ISO-induced cardiomyocyte shortening is about 78% on MI-N group, reduced to 28% in MI-IH and restored to 125% in MI-IH-CSD. Preliminary results on cardiac function indicate that CSD also improve long-term ejection fraction in MI-IH rats. Biochemical and histological analysis are on-going. These results suggest that IH-induced sympathetic activity is responsible for adrenergic reserve depletion in our rat model of ischemic cardiomyopathy. Ongoing experiments will confirm if CSD also limits the IH-induced cardiac remodeling and contractile dysfunction.

Isolation and Characterization of Intact Cardiomyocytes from Frozen and Fresh Human Myocardium and Mouse Hearts.

Procurement and characterization of intact human cells are essential for studies in regenerative medicine and translational medical research. The selection of the currently available approaches to isolate intact cells depends on the age of the hearts. To isolate cardiomyocytes from the fetal or neonatal myocardium, the myocardium can be minced into small tissue blocks followed by enzyme incubation. However, the fetal and neonatal cardiomyocytes are very soft and the morphology changes from long rod or spindle shape to spheres after isolation. Because of the dense packing of the cardiomyocytes and the strong cell-cell connection in adult myocardium, it is difficult to isolate the cardiomyocytes from adult myocardium by enzyme incubationonly. Aperfusion method is necessary to deliver the enzyme solution to the deep layers of the myocardium. However, intact hearts, which are very rare, are required for theperfusion method. Therefore, lacking methods to efficiently isolate cardiomyocytes from myocardium of various ages builds a barrier between basic research and clinical studies. Here, we describe a method for the isolation of intact cardiomyocytes from fresh or frozen human myocardium or fresh mouse hearts and the quantification of multinucleation, cardiomyocyte size, cell cycle activity, and total cardiomyocyte count per heart. We generalize this fixation-digestion method by isolating cells from a variety of mouse organs, including the liver, lung, and thymus.

Purification of Pluripotent Stem Cell-Derived Cardiomyocytes Using CRISPR/Cas9-Mediated Integration of Fluorescent Reporters.

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes have become critically important for the detailed study of cardiac development, disease modeling, and drug screening. However, directed differentiation of hiPSCs into cardiomyocytes often results in mixed populations of cardiomyocytes and other cell types, which may confound experiments that require pure populations of cardiomyocytes. Here, we detail the use of a CRISPR/Cas9 genome editing strategy to develop cardiomyocyte-specific reporters that allow for the isolation of hiPSC-derived cardiomyocytes and chamber-specific myocytes. Moreover, we describe a cardiac differentiation protocol to derive cardiomyocytes from hiPSCs, as well as a strategy to use fluorescence-activated cell sorting to isolate pure populations of fluorescently labeled cardiomyocytes for downstream applications.

Isolation, Culture, and Live-Cell Imaging of Primary Rat Cardiomyocytes.

The heart is a complex organ consisting of a variety of different cardiomyocytes (ventricular vs. atrial, left vs. right ventricular, working vs. nodal) as well as other cell types, including endothelial cells and vascular smooth muscle cells. Pericytes, neurons, and immune cells are less abundant, yet still important. Whereas cardiomyocytes account for around 75% of the heart volume, 50-70% of the cells in the heart are non-myocytes. This complexity of the heart underlines the difficulties in interpreting data obtained in vivo. In the field of cardiac regeneration, it remains unclear whether it is possible to induce a significant number of cardiomyocytes to proliferate and whether the often-observed improvement in cardiac function after experimental therapies is due to the induction of cardiomyocyte proliferation. Therefore, the reductionist approach inherent to cultures of isolated cells continues to be of great importance, even though it is important to study heart disease in vivo due to interactions of the different cell types. Cultured cardiomyocytes allow for easy manipulation of cell behavior (e.g., cell division) and its analysis (e.g., live-cell imaging). In addition, isolated cells in culture are a valuable tool for pharmacological and toxicological studies. This chapter offers a practical guide to isolate and culture primary neonatal and adult rat cardiomyocytes and a detailed protocol for live-cell imaging of embryonic and neonatal cardiomyocytes.

Approaches to High-Throughput Analysis of Cardiomyocyte Contractility.

The measurement of the contractile behavior of single cardiomyocytes has made a significant contribution to our understanding of the physiology and pathophysiology of the myocardium. However, the isolation of cardiomyocytes introduces various technical and statistical issues. Traditional video and fluorescence microscopy techniques based around conventional microscopy systems result in low-throughput experimental studies, in which single cells are studied over the course of a pharmacological or physiological intervention. We describe a new approach to these experiments made possible with a new piece of instrumentation, the CytoCypher High-Throughput System (CC-HTS). We can assess the shortening of sarcomeres, cell length, Ca2+ handling, and cellular morphology of almost 4 cells per minute. This increase in productivity means that batch-to-batch variation can be identified as a major source of variability. The speed of acquisition means that sufficient numbers of cells in each preparation can be assessed for multiple conditions reducing these batch effects. We demonstrate the different temporal scales over which the CC-HTS can acquire data. We use statistical analysis methods that compensate for the hierarchical effects of clustering within heart preparations and demonstrate a significant false-positive rate, which is potentially present in conventional studies. We demonstrate a more stringent way to perform these tests. The baseline morphological and functional characteristics of rat, mouse, guinea pig, and human cells are explored. Finally, we show data from concentration response experiments revealing the usefulness of the CC-HTS in such studies. We specifically focus on the effects of agents that directly or indirectly affect the activity of the motor proteins involved in the production of cardiomyocyte contraction. A variety of myocardial preparations with differing levels of complexity are in use (e.g., isolated muscle bundles, thin slices, perfused dual innervated isolated heart, and perfused ventricular wedge). All suffer from low throughput but can be regarded as providing independent data points in contrast to the clustering problems associated with isolated cell studies. The greater productivity and sampling power provided by CC-HTS may help to reestablish the utility of isolated cell studies, while preserving the unique insights provided by studying the contribution of the fundamental, cellular unit of myocardial contractility.

An improved two-step method for extraction and purification of primary cardiomyocytes from neonatal mice

IntroductionPrimary mouse cardiomyocytes are essential tools for cardiovascular pharmacology research at the cellular and molecular levels, but their low viability and low purity have often caused challenges in previous studies. Hence, we developed an improved two-step method for extraction and purification of primary cardiomyocytes from neonatal mice. MethodThis method consisted of two steps: 1) isolation and pre-digestion of heart tissues from 1- to 3-day-old C57 neonatal mice and 2) extraction and purification of cardiomyocytes. The traditional method of primary mouse cardiomyocyte isolation was used as the control group to assess the extraction efficiency of cardiomyocytes by the two-step method, and the purity and viability of cardiomyocytes were evaluated by immunofluorescence staining and autonomous beating analysis, respectively. ResultsCompared with the control method, the two-step method enabled acquisition of more cells from mouse hearts (1.28 ± 0.11 × 106vs 0.59 ± 0.15 × 106 cells/heart), and the resulting cells exhibited higher adherence rates and cell purity (93.25 ± 1.69% vs 73.62 ± 9.76%) after 48 h of culture. Moreover, the viability of cardiomyocytes was also evidently higher in the two-step group than in the control group (124.67 ± 10.50 vs 88.50 ± 6.61 beats/min). DiscussionCompared with the traditional method, the two-step method exhibited significantly better efficiency in extraction of primary cardiomyocytes and yielded cells with greater purity and viability. The two-step method will likely become a standard method for studies based on primary mouse cardiomyocytes in the future.

Human Heart Cardiomyocytes in Drug Discovery and Research: New Opportunities in Translational Sciences.

In preclinical drug development, accurate prediction of drug effects on the human heart is critically important, whether in the context of cardiovascular safety or for the purpose of modulating cardiac function to treat heart disease. Current strategies have significant limitations, whereby, cardiotoxic drugs can escape detection or potential life-saving therapies are abandoned due to false positive toxicity signals. Thus, new and more reliable translational approaches are urgently needed to help accelerate the rate of new therapy development. Renewed efforts in the recovery of human donor hearts for research and in cardiomyocyte isolation methods, are providing new opportunities for preclinical studies in adult primary cardiomyocytes. These cells exhibit the native physiological and pharmacological properties, overcoming the limitations presented by artificial cellular models, animal models and have great potential for providing an excellent tool for preclinical drug testing. Adult human primary cardiomyocytes have already shown utility in assessing drug-induced cardiotoxicity risk and helping in the identification of new treatments for cardiac diseases, such as heart failure and atrial fibrillation. Finally, strategies with actionable decision-making trees that rely on data derived from adult human primary cardiomyocytes will provide the holistic insights necessary to accurately predict human heart effects of drugs.