Marcel A.G. van der Heyden

TGF-β1 induces efficient differentiation of human cardiomyocyte progenitor cells into functional cardiomyocytes in vitro

The adult mammalian heart has limited regenerative capacity and was generally considered to contain no dividing cells. Recently, however, a resident population of progenitor cells has been identified, which could represent a new source of cardiomyocytes. Here, we describe the efficient isolation and propagation of human cardiomyocyte progenitor cells (hCMPCs) from fetal heart and patient biopsies. Establishment of hCMPC cultures was remarkably reproducible, with over 70% of adult atrial biopsies resulting in robustly expanding cell populations. Following the addition of transforming growth factor beta, almost all cells differentiated into spontaneously beating myocytes with characteristic cross striations. hCMPC-derived cardiomyocytes showed gap-junctional communication and action potentials of maturing cardiomyocytes. These are the first cells isolated from human heart that proliferate and form functional cardiomyocytes without requiring coculture with neonatal myocytes. Their scalability and homogeneity are unique and provide an excellent basis for developing physiological, pharmacological, and toxicological assays on human heart cells in vitro.

Twenty one years of P19 cells: what an embryonal carcinoma cell line taught us about cardiomyocyte differentiation

Many different stem cell types have been shown to differentiate into cardiac muscle cells in vitro but P19 embryonal carcinoma (EC) cells were one of the first examples described and have been the most extensively characterized. P19 EC cells, isolated from an experimental embryo-derived teratocarcinoma in mice, are multipotent and can differentiate into cell types of all three germ layers. Because of their capacity to form cardiomyocytes however, they have been used to dissect the role of cardiac-specific transcription factors and upstream signalling pathways in cardiac cell differentiation. Furthermore, they have shed light on unique aspects of cardiac cell physiology during heart cell differentiation, including regulation of the proteins underlying the electrical and contractile systems. Here, we review studies on different subclones of P19 cells, and what they have taught us about cardiac differentiation and physiology.

Cardiac cell-cell junctions in health and disease: Electrical versus mechanical coupling

Intercalated discs are the membrane sites where individual cardiomyocytes are connected to each other. Adherens-, desmosomal-, and gap junctions are situated in the intercalated disc and ensure mechanical coupling between cells and enable propagation of electrical impulses throughout the heart. A number of cardiac disorders, for example arrhythmogenic right ventricular dysplasia/cardiomyopathy, have been described in which an impaired mechanical coupling leads to electrical dysfunction, with occurrence of fatal arrhythmias. In this article the interaction between electrical and mechanical coupling is explored by reviewing studies performed in patients, animals, and in vitro. In these studies the effect of changes in protein composition of a mechanical junction on the electrical junction, and vice versa were investigated. It is shown that impaired electrical coupling does not change mechanical coupling. However, impaired mechanical coupling largely affects electrical coupling.

Dominant missense mutations in ABCC9 cause Cantú syndrome

Cantú syndrome is characterized by congenital hypertrichosis, distinctive facial features, osteochondrodysplasia and cardiac defects. By using family-based exome sequencing, we identified a de novo mutation in ABCC9. Subsequently, we discovered novel dominant missense mutations in ABCC9 in 14 of the 16 individuals with Cantú syndrome examined. The ABCC9 protein is part of an ATP-dependent potassium (KATP) channel that couples the metabolic state of a cell with its electrical activity. All mutations altered amino acids in or close to the transmembrane domains of ABCC9. Using electrophysiological measurements, we show that mutations in ABCC9 reduce the ATP-mediated potassium channel inhibition, resulting in channel opening. Moreover, similarities between the phenotype of individuals with Cantú syndrome and side effects from the KATP channel agonist minoxidil indicate that the mutations in ABCC9 result in channel opening. Given the availability of ABCC9 antagonists, our findings may have direct implications for the treatment of individuals with Cantú syndrome.

Remodeling of the cardiac sodium channel, connexin43, and plakoglobin at the intercalated disk in patients with arrhythmogenic cardiomyopathy

BACKGROUND Arrhythmogenic cardiomyopathy (AC) is closely associated with desmosomal mutations in a majority of patients. Arrhythmogenesis in patients with AC is likely related to remodeling of cardiac gap junctions and increased levels of fibrosis. Recently, using experimental models, we also identified sodium channel dysfunction secondary to desmosomal dysfunction. OBJECTIVE To assess the immunoreactive signal levels of the sodium channel protein NaV1.5, as well as connexin43 (Cx43) and plakoglobin (PKG), in myocardial specimens obtained from patients with AC. METHODS Left and right ventricular free wall postmortem material was obtained from 5 patients with AC and 5 controls matched for age and sex. Right ventricular septal biopsies were taken from another 15 patients with AC. All patients fulfilled the 2010 revised Task Force Criteria for the diagnosis of AC. Immunohistochemical analyses were performed using antibodies against Cx43, PKG, NaV1.5, plakophilin-2, and N-cadherin. RESULTS N-cadherin and desmoplakin immunoreactive signals and distribution were normal in patients with AC compared to controls. Plakophilin-2 signals were unaffected unless a plakophilin-2 mutation predicting haploinsufficiency was present. Distribution was unchanged compared to that in controls. Immunoreactive signal levels of PKG, Cx43, and NaV1.5 were disturbed in 74%, 70%, and 65% of the patients, respectively. CONCLUSIONS A reduced immunoreactive signal of PKG, Cx43, and NaV1.5 at the intercalated disks can be observed in a large majority of the patients. Decreased levels of Nav1.5 might contribute to arrhythmia vulnerability and, in the future, potentially could serve as a new clinically relevant tool for risk assessment strategies.

P19 embryonal carcinoma cells: a suitable model system for cardiac electrophysiological differentiation at the molecular and functional level

Objective: Murine P19 embryonal carcinoma (EC) cells can differentiate into spontaneously beating cardiomyocytes in vitro and have revealed important insight into the early molecular processes of cardiomyocyte differentiation. We assessed the suitability of the P19 cell model for studying cardiac ion channel regulation at the molecular and functional level. Methods: P19 cells were induced to differentiate towards cardiomyocytes. mRNAs for cardiac markers and ion channels were determined by RT-PCR at six timepoints during the differentiation process. Action potentials and individual ion currents were measured by whole cell patch clamp. Results: Ion channel mRNA expression of several channels is temporally regulated during differentiation, while others show little or no regulation. L-type calcium and transient outward channels are expressed from very early on, while sodium and delayed and inward rectifier channels are upregulated at somewhat later stages during differentiation, which mirrors the in vivo murine cardiomyocyte differentiation during embryogenesis. Spontaneous cardiomyocyte action potentials exhibit a low upstroke velocity, which often can be enhanced by hyperpolarizing the cells, hence activating thusfar dormant ion channels to contribute to the action potential upstroke. Action potential duration decreases considerably during the differentiation of spontaneously beating cells. In late stages, non-beating myocytes can be found which only generate action potentials upon electrical stimulation. Their shape is comparable to neonatal/juvenile ventricular mouse myocytes in culture. Finally, we show that P19-derived cardiomyocytes display a very complete set of functional ion channels. Conclusion: P19 cells represent a powerful model to study the regulation of myocardial electrophysiological differentiation at the molecular and functional level.

Grayanotoxin poisoning: 'mad honey disease' and beyond.

Many plants of the Ericaceae family, Rhododendron, Pieris, Agarista and Kalmia, contain diterpene grayanotoxins. Consumption of grayanotoxin containing leaves, flowers or secondary products as honey may result in intoxication specifically characterized by dizziness, hypotension and atrial-ventricular block. Symptoms are caused by an inability to inactivate neural sodium ion channels resulting in continuous increased vagal tone. Grayanotoxin containing products are currently sold online, which may pose an increasing risk. In humans, intoxication is rarely lethal, in contrast to cattle and pet poisoning cases. Scientific evidence for the medicinal properties of grayanotoxin containing preparations, such as honey or herbal preparation in use in folk medicine, is scarce, and such use may even be harmful.

Epigenetics: DNA demethylation promotes skeletal myotube maturation

Mesenchymal progenitor cells can be differentiated in vitro into myotubes that exhibit many characteristic features of primary mammalian skeletal muscle fibers. However, in general, they do not show the functional excitation‐contraction coupling or the striated sarcomere arrangement typical of mature myofibers. Epigenetic modifications have been shown to play a key role in regulating the progressional changes in transcription necessary for muscle differentiation. In this study, we demonstrate that treatment of murine C2C12 mesenchymal progenitor cells with 10 μM of the DNA methylation inhibitor 5‐azacytidine (5AC) promotes myogenesis, resulting in myotubes with enhanced maturity as compared to untreated myotubes. Specifically, 5AC treatment resulted in the up‐regulation of muscle genes at the myoblast stage, while at later stages nearly 50% of the 5AC‐treated myotubes displayed a mature, well‐defined sarcomere organization, as well as spontaneous contractions that coincided with action potentials and intracellular calcium transients. Both the percentage of striated myotubes and their contractile activity could be inhibited by 20 nM TTX, 10 μM ryanodine, and 100 μM nifedipine, suggesting that action potential‐induced calcium transients are responsible for these characteristics. Our data suggest that genomic demethylation induced by 5AC overcomes an epigenetic barrier that prevents untreated C2C12 myotubes from reaching full maturity.—Hupkes, M., Jonsson, M. K. B., Scheenen, W. J., van Rotterdam, W., Sotoca, A. M., van Someren, E. P., van der Heyden, M. A. G., van Veen, T. A., van Ravestein‐van Os, R. I., Bauerschmidt, S., Piek, E., Ypey, D. L., van Zoelen, E. J., Dechering, K. J. Epigenetics: DNA demethylation promotes skeletal myotube maturation. FASEB J. 25, 3861–3872 (2011). www.fasebj.org

Beat-to-beat variability of repolarization as a new biomarker for proarrhythmia in vivo

Pharmacological safety evaluation of (pro) drugs includes cardiac safety assessment of proarrhythmic liability in healthy tissue with emphasis on the rapid component of the delayed rectifier (I(Kr)). The lack of (1) an arrhythmic end point, (2) tests in remodeled, predisposed tissue, and (3) testing chronic drug influence on channel trafficking impairs on the drawn conclusions of these assays regarding drug safety. Moreover, the currently used human ether-à-go-go-related gene assays, action potential duration, prolongation in multicellular preparations, or the QT interval have significant shortcomings in their prediction of an increased risk for drug-induced torsades de pointes arrhythmia. In this review, it will be proposed that beat-to-beat variability of repolarization quantified as short-term variability can (1) discriminate between safe and unsafe drugs even under predisposed and highly arrhythmogenic conditions despite accompanying QT prolongation and (2) identify the individual at risk for subsequent arrhythmic events.

Expression of the Electrophysiological System During Murine Embryonic Stem Cell Cardiac Differentiation

Background: Stem cell based replacement therapy is envisioned as a method to repair failing hearts suffering from cardiomyocyte loss. To prevent potentially lethal arrhythmias, the donor cellular electrophysiological make-up should match with the acceptor tissue. To engineer the desired electrophysiological phenotype, the underlying molecular regulation of ion channels and gap-junction proteins should be clarified first. Methods: We established the expression of seven main cardiac ion channel a-subunits and four b-subunits using semiquantitive RT-PCR and two major cardiac gap-junction proteins by immunohistochemistry during the differentiation process of murine ES cells into cardiomyocytes. Results: Ion channel mRNA expression profiles display sequential upregulation. Connexin-40 and -43 expression is low in early cardiomyocytes, increased expression rates were found in subsequent differentiation phases. Conclusion: Cardiac differentiation of mouse embryonic stem cells is characterized by a sequential upregulation of the main cardiac ion channels and connexins.