A protocol for rapid and parallel isolation of myocytes and non-myocytes from multiple mouse hearts.

This protocol features parallel isolation of myocytes and non-myocytes from murine hearts. It was designed with considerations for (1) time required to extract cardiac cells, (2) cell viability, and (3) protocol scalability. Here, a peristaltic pump and 3D-printed elements are combined to perfuse the heart with enzymes to dissociate cells. Myocytes and non-myocytes extracted using this protocol are separated by centrifugation and/or fluorescence-activated cell sorting for use in downstream applications including single-cell omics or other bio-molecular analyses. For complete details on the use and execution of this protocol, please refer to McLellan et al. (2020).

Cells of the adult human heart.

Cardiovascular disease is the leading cause of death worldwide. Advanced insights into disease mechanisms and therapeutic strategies require a deeper understanding of the molecular processes involved in the healthy heart. Knowledge of the full repertoire of cardiac cells and their gene expression profiles is a fundamental first step in this endeavour. Here, using state-of-the-art analyses of large-scale single-cell and single-nucleus transcriptomes, we characterize six anatomical adult heart regions. Our results highlight the cellular heterogeneity of cardiomyocytes, pericytes and fibroblasts, and reveal distinct atrial and ventricular subsets of cells with diverse developmental origins and specialized properties. We define the complexity of the cardiac vasculature and its changes along the arterio-venous axis. In the immune compartment, we identify cardiac-resident macrophages with inflammatory and protective transcriptional signatures. Furthermore, analyses of cell-to-cell interactions highlight different networks of macrophages, fibroblasts and cardiomyocytes between atria and ventricles that are distinct from those of skeletal muscle. Our human cardiac cell atlas improves our understanding of the human heart and provides a valuable reference for future studies.

A computational model of induced pluripotent stem-cell derived cardiomyocytes for high throughput risk stratification of KCNQ1 genetic variants.

In the last decade, there has been tremendous progress in identifying genetic anomalies linked to clinical disease. New experimental platforms have connected genetic variants to mechanisms underlying disruption of cellular and organ behavior and the emergence of proarrhythmic cardiac phenotypes. The development of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) signifies an important advance in the study of genetic disease in a patient-specific context. However, considerable limitations of iPSC-CM technologies have not been addressed: 1) phenotypic variability in apparently identical genotype perturbations, 2) low-throughput electrophysiological measurements, and 3) an immature phenotype which may impact translation to adult cardiac response. We have developed a computational approach intended to address these problems. We applied our recent iPSC-CM computational model to predict the proarrhythmic risk of 40 KCNQ1 genetic variants. An IKs computational model was fit to experimental data for each mutation, and the impact of each mutation was simulated in a population of iPSC-CM models. Using a test set of 15 KCNQ1 mutations with known clinical long QT phenotypes, we developed a method to stratify the effects of KCNQ1 mutations based on proarrhythmic markers. We utilized this method to predict the severity of the remaining 25 KCNQ1 mutations with unknown clinical significance. Tremendous phenotypic variability was observed in the iPSC-CM model population following mutant perturbations. A key novelty is our reporting of the impact of individual KCNQ1 mutant models on adult ventricular cardiomyocyte electrophysiology, allowing for prediction of mutant impact across the continuum of aging. This serves as a first step toward translating predicted response in the iPSC-CM model to predicted response of the adult ventricular myocyte given the same genetic mutation. As a whole, this study presents a new computational framework that serves as a high throughput method to evaluate risk of genetic mutations based-on proarrhythmic behavior in phenotypically variable populations.

The neuropeptide GsMTx4 inhibits a mechanosensitive BK channel through the voltage-dependent modification specific to mechano-gating.

The cardiac mechanosensitive BK (Slo1) channels are gated by Ca2+, voltage, and membrane stretch. The neuropeptide GsMTx4 is a selective inhibitor of mechanosensitive (MS) channels. It has been reported to suppress stretch-induced cardiac fibrillation in the heart, but the mechanism underlying the specificity and even the targeting channel(s) in the heart remain elusive. Here, we report that GsMTx4 inhibits a stretch-activated BK channel (SAKcaC) in the heart through a modulation specific to mechano-gating. We show that membrane stretching increases while GsMTx4 decreases the open probability (Po) of SAKcaC. These effects were mostly abolished by the deletion of the STREX axis-regulated (STREX) exon located between RCK1 and RCK2 domains in BK channels. Single-channel kinetics analysis revealed that membrane stretch activates SAKcaC by prolonging the open-time duration (τO) and shortening the closed-time constant (τC). In contrast, GsMTx4 reversed the effects of membrane stretch, suggesting that GsMTx4 inhibits SAKcaC activity by interfering with mechano-gating of the channel. Moreover, GsMTx4 exerted stronger efficacy on SAKcaC under membrane-hyperpolarized/resting conditions. Molecular dynamics simulation study revealed that GsMTx4 appeared to have the ability to penetrate deeply within the bilayer, thus generating strong membrane deformation under the hyperpolarizing/resting conditions. Immunostaining results indicate that BK variants containing STREX are also expressed in mouse ventricular cardiomyocytes. Our results provide common mechanisms of peptide actions on MS channels and may give clues to therapeutic suppression of cardiac arrhythmias caused by excitatory currents through MS channels under hyper-mechanical stress in the heart.

Evidence for hormonal control of heart regenerative capacity during endothermy acquisition.

Tissue regenerative potential displays striking divergence across phylogeny and ontogeny, but the underlying mechanisms remain enigmatic. Loss of mammalian cardiac regenerative potential correlates with cardiomyocyte cell-cycle arrest and polyploidization as well as the development of postnatal endothermy. We reveal that diploid cardiomyocyte abundance across 41 species conforms to Kleiber's law-the ¾-power law scaling of metabolism with bodyweight-and inversely correlates with standard metabolic rate, body temperature, and serum thyroxine level. Inactivation of thyroid hormone signaling reduces mouse cardiomyocyte polyploidization, delays cell-cycle exit, and retains cardiac regenerative potential in adults. Conversely, exogenous thyroid hormones inhibit zebrafish heart regeneration. Thus, our findings suggest that loss of heart regenerative capacity in adult mammals is triggered by increasing thyroid hormones and may be a trade-off for the acquisition of endothermy.

Representation of Multiple Cellular Phenotypes Within Tissue-Level Simulations of Cardiac Electrophysiology.

Distinct electrophysiological phenotypes are exhibited by biological cells that have differentiated into particular cell types. The usual approach when simulating the cardiac electrophysiology of tissue that includes different cell types is to model the different cell types as occupying spatially distinct yet coupled regions. Instead, we model the electrophysiology of well-mixed cells by using homogenisation to derive an extension to the commonly used monodomain or bidomain equations. These new equations permit spatial variations in the distribution of the different subtypes of cells and will reduce the computational demands of solving the governing equations. We validate the homogenisation computationally, and then use the new model to explain some experimental observations from stem cell-derived cardiomyocyte monolayers.

3D bioprinted functional and contractile cardiac tissue constructs.

Bioengineering of a functional cardiac tissue composed of primary cardiomyocytes has great potential for myocardial regeneration and in vitro tissue modeling. However, its applications remain limited because the cardiac tissue is a highly organized structure with unique physiologic, biomechanical, and electrical properties. In this study, we undertook a proof-of-concept study to develop a contractile cardiac tissue with cellular organization, uniformity, and scalability by using three-dimensional (3D) bioprinting strategy. Primary cardiomyocytes were isolated from infant rat hearts and suspended in a fibrin-based bioink to determine the priting capability for cardiac tissue engineering. This cell-laden hydrogel was sequentially printed with a sacrificial hydrogel and a supporting polymeric frame through a 300-µm nozzle by pressured air. Bioprinted cardiac tissue constructs had a spontaneous synchronous contraction in culture, implying in vitro cardiac tissue development and maturation. Progressive cardiac tissue development was confirmed by immunostaining for α-actinin and connexin 43, indicating that cardiac tissues were formed with uniformly aligned, dense, and electromechanically coupled cardiac cells. These constructs exhibited physiologic responses to known cardiac drugs regarding beating frequency and contraction forces. In addition, Notch signaling blockade significantly accelerated development and maturation of bioprinted cardiac tissues. Our results demonstrated the feasibility of bioprinting functional cardiac tissues that could be used for tissue engineering applications and pharmaceutical purposes. STATEMENT OF SIGNIFICANCE: Cardiovascular disease remains a leading cause of death in the United States and a major health-care burden. Myocardial infarction (MI) is a main cause of death in cardiovascular diseases. MI occurs as a consequence of sudden blocking of blood vessels supplying the heart. When occlusions in the coronary arteries occur, an immediate decrease in nutrient and oxygen supply to the cardiac muscle, resulting in permanent cardiac cell death. Eventually, scar tissue formed in the damaged cardiac muscle that cannot conduct electrical or mechanical stimuli thus leading to a reduction in the pumping efficiency of the heart. The therapeutic options available for end-stage heart failure is to undergo heart transplantation or the use of mechanical ventricular assist devices (VADs). However, many patients die while being on a waiting list, due to the organ shortage and limitation of VADs, such as surgical complications, infection, thrombogenesis, and failure of the electrical motor and hemolysis. Ultimately, 3D bioprinting strategy aims to create clinically applicable tissue constructs that can be immediately implanted in the body. To date, the focus on replicating complex and heterogeneous tissue constructs continues to increase as 3D bioprinting technologies advance. In this study, we demonstrated the feasibility of 3D bioprinting strategy to bioengineer the functional cardiac tissue that possesses a highly organized structure with unique physiological and biomechanical properties similar to native cardiac tissue. This bioprinting strategy has great potential to precisely generate functional cardiac tissues for use in pharmaceutical and regenerative medicine applications.

Subtype-specific differentiation of cardiac pacemaker cell clusters from human induced pluripotent stem cells.

BACKGROUND: Human induced pluripotent stem cells (hiPSC) harbor the potential to differentiate into diverse cardiac cell types. Previous experimental efforts were primarily directed at the generation of hiPSC-derived cells with ventricular cardiomyocyte characteristics. Aiming at a straightforward approach for pacemaker cell modeling and replacement, we sought to selectively differentiate cells with nodal-type properties. METHODS: hiPSC were differentiated into spontaneously beating clusters by co-culturing with visceral endoderm-like cells in a serum-free medium. Subsequent culturing in a specified fetal bovine serum (FBS)-enriched cell medium produced a pacemaker-type phenotype that was studied in detail using quantitative real-time polymerase chain reaction (qRT-PCR), immunocytochemistry, and patch-clamp electrophysiology. Further investigations comprised pharmacological stimulations and co-culturing with neonatal cardiomyocytes. RESULTS: hiPSC co-cultured in a serum-free medium with the visceral endoderm-like cell line END-2 produced spontaneously beating clusters after 10-12 days of culture. The pacemaker-specific genes HCN4, TBX3, and TBX18 were abundantly expressed at this early developmental stage, while levels of sarcomeric gene products remained low. We observed that working-type cardiomyogenic differentiation can be suppressed by transfer of early clusters into a FBS-enriched cell medium immediately after beating onset. After 6 weeks under these conditions, sinoatrial node (SAN) hallmark genes remained at high levels, while working-type myocardial transcripts (NKX2.5, TBX5) were low. Clusters were characterized by regular activity and robust beating rates (70-90 beats/min) and were triggered by spontaneous Ca2+ transients recapitulating calcium clock properties of genuine pacemaker cells. They were responsive to adrenergic/cholinergic stimulation and able to pace neonatal rat ventricular myocytes in co-culture experiments. Action potential (AP) measurements of cells individualized from clusters exhibited nodal-type (63.4%) and atrial-type (36.6%) AP morphologies, while ventricular AP configurations were not observed. CONCLUSION: We provide a novel culture media-based, transgene-free approach for targeted generation of hiPSC-derived pacemaker-type cells that grow in clusters and offer the potential for disease modeling, drug testing, and individualized cell-based replacement therapy of the SAN.

Human Embryonic Stem Cell-Derived Cardiomyocytes Self-Arrange with Areas of Different Subtypes During Differentiation.

The derivation of functional cardiomyocytes (CMs) from human embryonic stem cells (hESCs) represents a unique way of studying human cardiogenesis, including the development of CM subtypes. In this study, we investigated the development and organization of hESC-derived cardiomyocytes (hESC-CMs) and examined how the expression levels of CM subtypes correspond to human in vivo cardiogenesis. Beating clusters were used to determine cardiac differentiation, which was evaluated by the expression of cardiac genes GATA4 and TNNT2 and subcellular localization of GATA4 and NKX2.5. Sharp electrode recordings to determine action potentials (APs) further revealed spatial organization of intracluster CM subtypes (ie, complex clusters). Nodal-, atrial-, and ventricular-like AP morphologies were detected within distinct regions of complex clusters. The ability of different CM subtypes to self-organize was documented by immunohistochemical analyses and a differential spatial expression of ß-III tubulin, myosin light chain 2v (MLC-2V), and α-smooth muscle actin (α-SMA). Furthermore, all hESC-CM subtypes formed expressed primary cilia, which are known to coordinate cellular signaling pathways during cardiomyogenesis and heart development. This study expands the foundation for studying regulatory pathways for spatial and temporal CM differentiation during human cardiogenesis.

Semi-automated program for analysis of local Ca2+ spark release with application for classification of heart cell type.

Local Ca2+ spark releases are essential to the Ca2+ cycling process. Thus, they play an important role in ventricular and atrial cell contraction, as well as in sinoatrial cell automaticity. Characterizing their properties in healthy cells from different regions in the heart can reveal the basic biophysical differences among these regions. We designed a semi-automatic Matlab Graphical User Interface (called Sparkalyzer) to characterize parameters of Ca2+ spark release from any major cardiac tissue, as recorded in line-scan mode with a confocal laser-scanning microscope. We validated the algorithm on experimental images from rabbit sinoatrial, atrial, and ventricular cells loaded with Fluo-4 AM. The program characterizes general image parameters of Ca2+ transients and sparks: spark duration, which indicates for how long the spark provides Ca2+ to the closed intracellular mechanisms (typical value: 25±1, 23±1, 26±1ms for sinoatrial, atrial, and ventricular cells, respectively); spark amplitude, which indicates the amount of Ca2+ released by a single spark (1.6±0.1, 1.6±0.2, 1.4±0.1F/F0 for sinoatrial, atrial, and ventricular cells, respectively); spark length, which is the length of the Ca2+ wavelets fired out of a row of ryanodine receptors (5±0.1, 5±0.2, 3.4±0.3µm for sinoatrial, atrial, or ventricular cells, respectively) and number of sparks (0.14±0.02, 0.025±0.01, 0.02±0.01 for 1µm in 1s for sinoatrial, atrial, and ventricular cells, respectively). This method is reliable for Ca2+ spark analysis of sinoatrial, atrial, or ventricular cells. Moreover, by examining the average value of Ca2+ spark characteristics and their scattering around the mean, atrial, ventricular and sinoatrial cells can be differentiated.

Human induced pluripotent stem cell-derived cardiomyocytes: insights into molecular, cellular, and functional phenotypes.

Disease models are essential for understanding cardiovascular disease pathogenesis and developing new therapeutics. The human induced pluripotent stem cell (iPSC) technology has generated significant enthusiasm for its potential application in basic and translational cardiac research. Patient-specific iPSC-derived cardiomyocytes offer an attractive experimental platform to model cardiovascular diseases, study the earliest stages of human development, accelerate predictive drug toxicology tests, and advance potential regenerative therapies. Harnessing the power of iPSC-derived cardiomyocytes could eliminate confounding species-specific and interpersonal variations and ultimately pave the way for the development of personalized medicine for cardiovascular diseases. However, the predictive power of iPSC-derived cardiomyocytes as a valuable model is contingent on comprehensive and rigorous molecular and functional characterization.

Growth characteristics of different heart cells on novel nanopatch substrate during electrical stimulation.

During a heart attack, the heart's oxygen supply is cut off, and cardiomyocytes perish. Unfortunately, once these tissues are lost, they cannot be replaced and results in cardiovascular disease-the leading cause of deaths worldwide. Advancements in medical research have been targeted to understand and combat the death of these cardiomyocytes. For example, new research (in vitro) has demonstrated that one can expand cardiomyocyte adhesion and proliferation using polylactic-co-glycolic acid (PLGA) (50:50 (weight percent)) supplemented with carbon nanofibers (CNFs) to create a cardiovascular patch. However, the examination of other cardiovascular cell types has not been investigated. Therefore, the purpose of this present in vitro study was to determine cell growth characteristics of three different important cardiovascular cell types (aortic endothelial, fibroblast and cardiomyocyte) onto the substrate. Cells were seeded onto different PLGA:CNF ratio composites to determine if CNF density has an effect on cell growth, both in static and electrically stimulated environments. During continuous electrical stimulation (rectangle, 2 nm, 5 V/cm, 1 Hz), cardiomyocyte cell density increased in comparison to its static counterparts after 24, 72 and 120 hours. A minor rise in Troponin I excretion in electrical stimulation compared to static conditions indicated nominal cardiomyocyte cell function during cell experiments. Endothelial and fibroblast cell growth experiments indicated the material hindered or stalled proliferation during both static and electrical stimulation experiments, thus supporting the growth of cardiomyocytes onto the dead tissue zone. Furthermore, the results specified that CNF density did have an effect on PLGA:CNF composite cytocompatibility properties with the best results coming from the 50:50 [PLGA:CNF (weight percent:weight percent)] composite. Therefore, this study provides further evidence that a conductive scaffold using nanotechnology should be further research for various cardiovascular applications.

Human-induced pluripotent stem cell-derived cardiomyocytes exhibit temporal changes in phenotype.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) have been recently derived and are used for basic research, cardiotoxicity assessment, and phenotypic screening. However, the hiPS-CM phenotype is dependent on their derivation, age, and culture conditions, and there is disagreement as to what constitutes a functional hiPS-CM. The aim of the present study is to characterize the temporal changes in hiPS-CM phenotype by examining five determinants of cardiomyocyte function: gene expression, ion channel functionality, calcium cycling, metabolic activity, and responsiveness to cardioactive compounds. Based on both gene expression and electrophysiological properties, at day 30 of differentiation, hiPS-CMs are immature cells that, with time in culture, progressively develop a more mature phenotype without signs of dedifferentiation. This phenotype is characterized by adult-like gene expression patterns, action potentials exhibiting ventricular atrial and nodal properties, coordinated calcium cycling and beating, suggesting the formation of a functional syncytium. Pharmacological responses to pathological (endothelin-1), physiological (IGF-1), and autonomic (isoproterenol) stimuli similar to those characteristic of isolated adult cardiac myocytes are present in maturing hiPS-CMs. In addition, thyroid hormone treatment of hiPS-CMs attenuated the fetal gene expression in favor of a more adult-like pattern. Overall, hiPS-CMs progressively acquire functionality when maintained in culture for a prolonged period of time. The description of this evolving phenotype helps to identify optimal use of hiPS-CMs for a range of research applications.

Higher frequencies of BCRP+ cardiac resident cells in ischaemic human myocardium.

AIMS: Several cardiac resident progenitor cell types have been reported for the adult mammalian heart. Here we characterize their frequencies and distribution pattern in non-ischaemic human myocardial tissue and after ischaemic events. METHODS AND RESULTS: We obtained 55 biopsy samples from human atria and ventricles and used immunohistological analysis to investigate two cardiac cell types, characterized by the expression of breast cancer resistance protein (BCRP)/ABCG2 [for side population (SP) cells] or c-kit. Highest frequencies of BCRP+ cells were detected in the ischaemic right atria with a median of 5.40% (range: 2.48-11.1%) vs. 4.40% (1.79-7.75%) in the non-ischaemic right atria (P = 0.47). Significantly higher amounts were identified in ischaemic compared with non-ischaemic ventricles, viz. 5.44% (3.24-9.30%) vs. 0.74% (0-5.23%) (P = 0.016). Few numbers of BCRP+ cells co-expressed the cardiac markers titin, sarcomeric α-actinin, or Nkx2.5; no co-expression of BCRP and progenitor cell marker Sca-1 or pluripotency markers Oct-3/4, SSEA-3, and SSEA-4 was detected. C-kit+ cells displayed higher frequencies in ischaemic (ratio: 1:25 000 ± 2500 of cell counts) vs. non-ischaemic myocardium (1:105 000 ± 43 000). Breast cancer resistance protein+/c-kit+ cells were not identified. Following in vitro differentiation, BCRP+ cells isolated from human heart biopsy samples (n = 6) showed expression of cardiac troponin T and α-myosin heavy-chain, but no full differentiation into functional beating cardiomyocytes was observed. CONCLUSION: We were able to demonstrate that BCRP+/CD31- cells are more abundant in the heart than their c-kit+ counterparts. In the non-ischaemic hearts, they are preferentially located in the atria. Following ischaemia, their numbers are elevated significantly. Our data might provide a valuable snapshot at potential progenitor cells after acute ischaemia in vivo, and mapping of these easily accessible cells may influence future cell therapeutic strategies.

Cardiac myocytes' dynamic contractile behavior differs depending on heart segment.

Cardiac myocytes originating from different parts of the heart exhibit varying morphology and ultrastructure. However, the difference in their dynamic behavior is unclear. We examined the contraction of cardiac myocytes originating from the apex, ventricle, and atrium, and found that their dynamic behavior, such as amplitude and frequency of contraction, differs depending on the heart segment of origin. Using video microscopy and high-precision image correlation, we found that: (1) apex myocytes exhibited the highest contraction rate (∼17 beats/min); (2) ventricular myocytes exhibited the highest contraction amplitude (∼5.2 micron); and (3) as myocyte contraction synchronized, their frequency did not change significantly, but the amplitude of contraction increased in apex and ventricular myocytes. In addition, as myocyte cultures mature they formed contractile filaments, further emphasizing the difference in myocyte dynamics is persistent. These results suggest that the dynamic behavior (in addition to static properties) of myocytes is dependent on their segment of origin.

Neuregulin/ErbB signaling regulates cardiac subtype specification in differentiating human embryonic stem cells.

RATIONALE: Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) exhibit either a "working" chamber or a nodal-like phenotype. To generate optimal hESC-CM preparations for eventual clinical application in cell-based therapies, we will need to control their differentiation into these specialized cardiac subtypes. OBJECTIVE: To demonstrate intact neuregulin (NRG)-1ß/ErbB signaling in hESC-CMs and test the hypothesis that this signaling pathway regulates cardiac subtype abundance in hESC-CM cultures. METHODS AND RESULTS: All experiments used hESC-CM cultures generated using our recently reported directed differentiation protocol. To support subsequent action potential phenotyping approaches and provide a higher-throughput method of determining cardiac subtype, we first developed and validated a novel genetic label that identifies nodal-type hESC-CMs. Next, control hESC-CM preparations were compared to those differentiated in the presence of exogenous NRG-1ß, an anti-NRG-1ß neutralizing antibody, or the ErbB antagonist AG1478. We used 3 independent approaches to determine the ratio of cardiac subtypes in the resultant populations: direct action potential phenotyping under current-clamp, activation of the aforementioned genetic label, and subtype-specific marker expression by RT-PCR. Using all 3 end points, we found that inhibition of NRG-1ß/ErbB signaling greatly enhanced the proportion of cells showing the nodal phenotype. CONCLUSIONS: NRG-1ß/ErbB signaling regulates the ratio of nodal- to working-type cells in differentiating hESC-CM cultures and presumably functions similarly during early human heart development. We speculate that, by manipulating NRG-1ß/ErbB signaling, it will be possible to generate preparations of enriched working-type myocytes for infarct repair, or, conversely, nodal cells for potential use in a biological pacemaker.

Developmental basis for electrophysiological heterogeneity in the ventricular and outflow tract myocardium as a substrate for life-threatening ventricular arrhythmias.

Reentry is the main mechanism of life-threatening ventricular arrhythmias, including ventricular fibrillation and tachycardia. Its occurrence depends on the simultaneous presence of an arrhythmogenic substrate (a preexisting condition) and a "trigger," and is favored by electrophysiological heterogeneities. In the adult heart, electrophysiological heterogeneities of the ventricle exist along the apicobasal, left-right, and transmural axes. Also, conduction is preferentially slowed in the right ventricular outflow tract, especially during pharmacological sodium channel blockade. We propose that the origin of electrophysiological heterogeneities of the adult heart lies in early heart development. The heart is formed from several progenitor regions: the first heart field predominantly forms the left ventricle, whereas the second heart field forms the right ventricle and outflow tract. Furthermore, the embryonic outflow tract consists of slowly conducting tissue until it is incorporated into the ventricles and develops rapidly conducting properties. The subepicardial myocytes and subendocardial myocytes run distinctive gene programs from their formation onwards. This review discusses the hypothesis that electrophysiological heterogeneities in the adult heart result from persisting patterns in gene expression and function along the craniocaudal and epicardial-endocardial axes of the developing heart. Understanding the developmental origins of electrophysiological heterogeneity contributing to ventricular arrhythmias may give rise to new therapies.

Engineering cardiac tissue from embryonic stem cells.

Restoration of cardiac function by replacement of diseased myocardium with functional cardiac myocytes may offer a potential cure for cardiac disease and will likely revolutionize treatment methods. During the past 20 years, we have seen the development of tissue engineering; among these types of tissue engineering is cardiac tissue engineering. This type of cardiac tissue engineering includes growing neonatal cardiomyocytes on preformed polymers, liquid collagen, and temperature-responsive surfaces. It also includes the application of neonatal rat or chick cardiomyocytes to skeletal myoblasts, mesenchymal stem cells and embryonic stem cells, static culture, and bioreactor and stretching cultivation. Progress has come step-by-step, but, in recent years, with great technological advances, the progress has been accelerating, moving this area of research from dream to reality. The engineered cardiac tissue not only reproduces in vitro, but it can also be shaped so that it will, at some time, be able to form valves or endothelial lining. This chapter describes the currently used protocols for cardiac tissue engineering: liquid collagen-based cardiac tissue engineering and cell sheet-based cardiac tissue engineering, especially cardiac tissue engineering using cardiomyocytes derived from embryonic stem cells.

Identification and characterization of embryonic stem cell-derived pacemaker and atrial cardiomyocytes.

The aim of this study was to identify and functionally characterize cardiac subtypes during early stages of development. For this purpose, transgenic embryonic stem cells were generated using the alpha-myosin heavy chain promoter driving the expression of the enhanced green fluorescent protein (EGFP). EGFP-positive clusters of cells were first observed as early as 7 days of development, thus, even before the initiation of the contractile activity. Flow cytometry and single-cell fluorescence measurements evidenced large diversities of EGFP intensity. Patch-clamp experiments showed EGFP expression exclusively in pacemaker and atrial but not ventricular cells. The highest fluorescence intensities were detected in pacemaker-like cardiomyocytes. In accordance, multielectrode-array recordings of whole embryoid bodies confirmed that the pacemaker center coincided with strongly EGFP-positive areas. The cardiac subtypes displayed already at this early stage differential characteristics of electrical activity and ion channel expression. Thus, quantitation of the alpha-myosin heavy chain driven reporter gene expression allows identification and functional characterization of early cardiac subtypes.

Localization of NOS-1 in the sarcolemma region of a subpopulation of atrial cardiomyocytes including myoendocrine cells and NOS-3 in vascular and endocardial endothelial cells of the rat heart.

Cellular localization patterns of NOS isoforms 1 and 3 (nNOS and eNOS, respectively) in the mammalian heart under basal (non-stimulated) working conditions are still a matter of discussion. Therefore, this issue was reinvestigated in rats using RT-PCR, Western blotting, catalytic histochemistry, immunohistochemistry and image analysis. Tongue and extensor digitorum longus muscles served as positive controls for NOS-1 and NOS-3. RT-PCR revealed NOS-1 mRNA and NOS-3 mRNA in atria and ventricles. Western blotting showed NOS-1 protein in atria and NOS-3 protein in the walls of both heart chambers. Localization of the activity of urea-resistant (and therefore specific) NADPH diaphorase (NADPH-D) and NOS-1 immunohistochemistry showed that NOS-1 is present in the sarcolemma region of a subpopulation of atrial cardiomyocytes but not in working and impulse-conducting cardiomyocytes of atria and ventricles. Atrial natriuretic peptide (ANP) immunohistochemistry revealed that a minority of the NOS-1-expressing atrial cardiomyocytes are myoendocrine cells. eNOS immunostaining was present in endothelial cells of capillaries of the conducting and working myocardium and endocardial cells. Image analysis of the activity of urea-resistant NOS diaphorase showed that NOS-1 activity is lower in the sarcolemma region of atrial cardiomyocytes than in that of tongue and extensor digitorum longus myofibers. These data suggest that, in the non-stimulated rat heart. NOS-1 is expressed in a subpopulation of atrial cardiomyocytes including myoendocrine cells, and that NOS-3 is expressed in the vascular and endocardial endothelium.