Azra Fatima

In vitro Modeling of Ryanodine Receptor 2 Dysfunction Using Human Induced Pluripotent Stem Cells

Background/Aims: Induced pluripotent stem (iPS) cells generated from accessible adult cells of patients with genetic diseases open unprecedented opportunities for exploring the pathophysiology of human diseases in vitro. Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is an inherited cardiac disorder that is caused by mutations in the cardiac ryanodine receptor type 2 gene (RYR2) and is characterized by stress-induced ventricular arrhythmia that can lead to sudden cardiac death in young individuals. The aim of this study was to generate iPS cells from a patient with CPVT1 and determine whether iPS cell-derived cardiomyocytes carrying patient specific RYR2 mutation recapitulate the disease phenotype in vitro. Methods: iPS cells were derived from dermal fibroblasts of healthy donors and a patient with CPVT1 carrying the novel heterozygous autosomal dominant mutation p.F2483I in the RYR2. Functional properties of iPS cell derived-cardiomyocytes were analyzed by using whole-cell current and voltage clamp and calcium imaging techniques. Results: Patch-clamp recordings revealed arrhythmias and delayed afterdepolarizations (DADs) after catecholaminergic stimulation of CPVT1-iPS cell-derived cardiomyocytes. Calcium imaging studies showed that, compared to healthy cardiomyocytes, CPVT1-cardiomyocytes exhibit higher amplitudes and longer durations of spontaneous Ca2+ release events at basal state. In addition, in CPVT1-cardiomyocytes the Ca2+-induced Ca2+-release events continued after repolarization and were abolished by increasing the cytosolic cAMP levels with forskolin. Conclusion: This study demonstrates the suitability of iPS cells in modeling RYR2-related cardiac disorders in vitro and opens new opportunities for investigating the disease mechanism in vitro, developing new drugs, predicting their toxicity, and optimizing current treatment strategies.

Functional characterization of cardiomyocytes derived from murine induced pluripotent stem cells in vitro

Several types of terminally differentiated somatic cells can be reprogrammed into a pluripotent state by ectopic expression of Klf4, Oct3/4, Sox2, and c‐Myc. Such induced pluripotent stem (iPS) cells have great potential to serve as an autologous source of cells for tissue repair. In the process of developing iPS‐cell‐based therapies, the major goal is to determine whether differentiated cells derived from iPS cells, such as cardiomyocytes (CMs), have the same functional properties as their physiological in vivo counterparts. Therefore, we differentiated murine iPS cells to CMs in vitro and characterized them by RTPCR, immunocytochemistry, and electrophysiology. As key markers of cardiac lineages, transcripts for Nkx2.5, αMHC, Mlc2v, and cTnT could be identified. Immunocytochemical stainings revealed the presence of organized sarcomeric actinin but the absence of mature atrial natriuretic factor. We examined characteristics and developmental changes of action potentials, as well as functional hormonal regulation and sensitivity to channel blockers. In addition, we determined expression patterns and functionality of cardiac‐specific voltage‐gated Na+, Ca2+, and K+ channels at early and late differentiation stages and compared them with CMs derived from murine embryonic stem cells (ESCs) as well as with fetal CMs. We conclude that iPS cells give rise to functional CMs in vitro, with established hormonal regulation pathways and functionally expressed cardiac ion channels;CMs generated from iPS cells have a ventricular phenotype;and cardiac development of iPS cells is delayed compared with maturation of native fetal CMs and of ESC‐derived CMs. This difference may reflect the incomplete reprogramming of iPS cells and should be critically considered in further studies to clarify the suitability of the iPS model for regenerative medicine of heart disorders.—Kuzmenkin, A., Liang, H., Xu, G., Pfannkuche, K., Eichhorn, H., Fatima, A., Luo, H., Saric, T., Wernig, M., Jaenisch, R., Hescheler, J. Functional characterization of cardiomyocytes derived from murine induced pluripotent stem cells in vitro. FASEB J. 23, 4168‐4180 (2009). www.fasebj.org

Comparison of contractile behavior of native murine ventricular tissue and cardiomyocytes derived from embryonic or induced pluripotent stem cells

Cardiomyocytes generated from embryonic stem cells (ESCs) and induced pluripotent stem (iPS) cells are suggested for repopulation of destroyed myocardium. Because contractile properties are crucial for functional regeneration, we compared cardiomyocytes differentiated from ES cells (ESC‐CMs) and iPS cells (iPS‐CMs). Native myocardium served as control. Murine ESCs or iPS cells were differentiated 11 d in vitro and cocultured 5–7 d with irreversibly injured myocardial tissue slices. Vital embryonic ventricular tissue slices of similar age served for comparison. Force‐frequency relationship (FFR), effects of Ca2+, Ni2+, nifedipine, ryanodine, β‐adrenergic, and muscarinic modulation were studied during loaded contractions. FFR was negative for ESC‐CMs and iPS‐CMs. FFR was positive for embryonic tissue and turned negative after treatment with ryanodine. In all groups, force of contraction and relaxation time increased with the concentration of Ca2+ and decreased with nifedipine. Force was reduced by Ni2+. Isoproterenol (1 µM) increased the force most pronounced in embryonic tissue (207±31%, n=7;ESC‐CMs: 123±5%, n=4; iPS‐CMs: 120 ±4%, n=8). EC50 values were similar. Contractile properties of iPS‐CMs and ESC‐CMs were similar, but they were significantly different from ventricular tissue of comparable age. The results indicate immaturity of the sarcoplasmic reticulum and the β‐adrenergic response of iPS‐CMs and ESC‐CMs.—Xi, J., Khalil, M., Shishechian, N., Hannes, T., Pfannkuche, K., Liang, H., Fatima, A., Haustein, M., Suhr, F., Bloch, W., Reppel, M., Šarić, T., Wernig, M., Jaenisch, R., Brockmeier, K., Hescheler, J., Pillekamp, F. Comparison of contractile behavior of native murine ventricular tissue and cardiomyocytes derived from embryonic or induced pluripotent stem cells. FASEB J. 24, 2739–2751 (2010). www.fasebj.org

Cardiac myocytes derived from murine reprogrammed fibroblasts: intact hormonal regulation, cardiac ion channel expression and development of contractility.

Aims: Induced pluripotent stem (iPS) cells have a developmental potential similar to that of blastocyst-derived embryonic stem (ES) cells and may serve as an autologous source of cells for tissue repair, in vitro disease modelling and toxicity assays. Here we aimed at generating iPS cell-derived cardiomyocytes (CMs) and comparing their molecular and functional characteristics with CMs derived from native murine ES cells. Methods and Results: Beating cardiomyocytes were generated using a mass culture system from murine N10 and O9 iPS cells as well as R1 and D3 ES cells. Transcripts of the mesoderm specification factor T-brachyury and non-atrial cardiac specific genes were expressed in differentiating iPS EBs. Using immunocytochemistry to determine the expression and intracellular organisation of cardiac specific structural proteins we demonstrate strong similarity between iPS-CMs and ES-CMs. In line with a previous study electrophysiological analyses showed that hormonal response to β-adrenergic and muscarinic receptor stimulation was intact. Action potential (AP) recordings suggested that most iPS-CMs measured up to day 23 of differentiation are of ventricular-like type. Application of lidocaine, Cs+, SEA0400 and verapamil+ nifedipine to plated iPS-EBs during multi-electrode array (MEA) measurements of extracellular field potentials and intracellular sharp electrode recordings of APs revealed the presence of INa, If, INCX, and ICaL, respectively, and suggested their involvement in cardiac pacemaking, with ICaL being of major importance. Furthermore, iPS-CMs developed and conferred force to avitalized ventricular tissue that was responsive to β-adrenergic stimulation. Conclusions: Our data demonstrate that the cardiogenic potential of iPS cells is comparable to that of ES cells and that iPS-CMs possess all fundamental functional elements of a typical cardiac cell, including spontaneous beating, hormonal regulation, cardiac ion channel expression and contractility. Therefore, iPS-CMs can be regarded as a potentially valuable source of cells for in vitro studies and cellular cardiomyoplasty.

Ca2+ signaling in human induced pluripotent stem cell-derived cardiomyocytes (iPS-CM) from normal and catecholaminergic polymorphic ventricular tachycardia (CPVT)-afflicted subjects.

Derivation of cardiomyocytes from induced pluripotent stem cells (iPS-CMs) allowed us to probe the Ca(2+)-signaling parameters of human iPS-CMs from healthy- and catecholaminergic polymorphic ventricular tachycardia (CPVT1)-afflicted individuals carrying a novel point mutation p.F2483I in ryanodine receptors (RyR2). iPS-CMs were dissociated on day 30-40 of differentiation and patch-clamped within 3-6 days. Calcium currents (ICa) averaged ∼8pA/pF in control and mutant iPS-CMs. ICa-induced Ca(2+)-transients in control and mutant cells had bell-shaped voltage-dependence similar to that of ICa, consistent with Ca(2+)-induced Ca(2+)-release (CICR) mechanism. The ratio of ICa-activated to caffeine-triggered Ca(2+)-transients was ∼0.3 in both cell types. Caffeine-induced Ca(2+)-transients generated significantly smaller Na(+)-Ca(2+) exchanger current (INCX) in mutant cells, reflecting their smaller Ca(2+)-stores. The gain of CICR was voltage-dependent as in adult cardiomyocytes. Adrenergic agonists enhanced ICa, but differentially altered the CICR gain, diastolic Ca(2+), and Ca(2+)-sparks in mutant cells. The mutant cells, when Ca(2+)-overloaded, showed longer and wandering Ca(2+)-sparks that activated adjoining release sites, had larger CICR gain at -30mV yet smaller Ca(2+)-stores. We conclude that control and mutant iPS-CMs express the adult cardiomyocyte Ca(2+)-signaling phenotype. RyR2 F2483I mutant myocytes have aberrant unitary Ca(2+)-signaling, smaller Ca(2+)-stores, higher CICR gains, and sensitized adrenergic regulation, consistent with functionally altered Ca(2+)-release profile of CPVT syndrome.

The disease-specific phenotype in cardiomyocytes derived from induced pluripotent stem cells of two long QT syndrome type 3 patients.

Long QT syndromes (LQTS) are heritable diseases characterized by prolongation of the QT interval on an electrocardiogram, which often leads to syncope and sudden cardiac death. Here we report the generation of induced pluripotent stems (iPS) cells from two patients with LQTS type 3 carrying a different point mutation in a sodium channel Nav1.5 (p.V240M and p.R535Q) and functional characterization of cardiomyocytes (CM) derived from them. The iPS cells exhibited all characteristic properties of pluripotent stem cells, maintained the disease-specific mutation and readily differentiated to CM. The duration of action potentials at 50% and 90% repolarization was longer in LQTS-3 CM as compared to control CM but this difference did not reach statistical significance due to high variations among cells. Sodium current recordings demonstrated longer time to peak and longer time to 90% of inactivation of the Na+ channel in the LQTS-3 CM. This hints at a defective Na+ channel caused by deficiency in open-state inactivation of the Na+ channel that is characteristic of LQTS-3. These analyses suggest that the effect of channel mutation in the diseased CM is demonstrated in vitro and that the iPS cell-derived CM can serve as a model system for studying the pathophysiology of LQTS-3, toxicity testing and design of novel therapeutics. However, further improvements in the model are still required to reduce cell-to-cell and cell line-to-cell line variability.

Electrophysiological integration and action potential properties of transplanted cardiomyocytes derived from induced pluripotent stem cells

AIMS Induced pluripotent stem cell-derived cardiomyocytes (iPSCM) are regarded as promising cell type for cardiac cell replacement therapy. We investigated long-term electrophysiological integration and maturation of transplanted iPSCM, which are essential for therapeutic benefit. METHODS AND RESULTS Murine iPSCM expressing enhanced green fluorescent protein and a puromycin resistance under control of the α-myosin heavy chain promoter were purified by antibiotic selection and injected into adult mouse hearts. After 6-12 days, 3-6 weeks, or 6-8 months, viable slices of recipient hearts were prepared. Slices were focally stimulated by a unipolar electrode placed in host tissue, and intracellular action potentials (APs) were recorded with glass microelectrodes in transplanted cells and neighbouring host tissue within the slices. Persistence and electrical integration of transplanted iPSCM into recipient hearts could be demonstrated at all time points. Quality of coupling improved, as indicated by a maximal stimulation frequency without conduction blocks of 5.77 ± 0.54 Hz at 6-12 days, 8.98 ± 0.38 Hz at 3-6 weeks and 10.82 ± 1.07 Hz at 6-8 months after transplantation. AP properties of iPSCM became more mature from 6-12 days to 6-8 months after transplantation, but still differed significantly from those of host APs. CONCLUSION Transplanted iPSCM can persist in the long term and integrate electrically into host tissue, supporting their potential for cell replacement therapy. Quality of electrical integration improves between 6-12 days and 6-8 months after transplantation, and there are signs of an electrophysiological maturation. However, even after 6-8 months, AP properties of transplanted iPSCM differ from those of recipient cardiomyocytes.

Dual Color Photoactivation Localization Microscopy of Cardiomyopathy-associated Desmin Mutants

Background: Heterozygous DES mutations affect filament formation leading to skeletal and cardiomyopathies. Results: Our results reveal different extent of filament formation defects by various desmin mutants under heterozygous conditions. Conclusion: Analysis of interaction and co-localization of mutant and wild-type desmin proves the co-existence of heterogeneous filaments in living cells. Significance: These results might be of relevance for the understanding of filament formation defects. Mutations in the DES gene coding for the intermediate filament protein desmin may cause skeletal and cardiac myopathies, which are frequently characterized by cytoplasmic aggregates of desmin and associated proteins at the cellular level. By atomic force microscopy, we demonstrated filament formation defects of desmin mutants, associated with arrhythmogenic right ventricular cardiomyopathy. To understand the pathogenesis of this disease, it is essential to analyze desmin filament structures under conditions in which both healthy and mutant desmin are expressed at equimolar levels mimicking an in vivo situation. Here, we applied dual color photoactivation localization microscopy using photoactivatable fluorescent proteins genetically fused to desmin and characterized the heterozygous status in living cells lacking endogenous desmin. In addition, we applied fluorescence resonance energy transfer to unravel short distance structural patterns of desmin mutants in filaments. For the first time, we present consistent high resolution data on the structural effects of five heterozygous desmin mutations on filament formation in vitro and in living cells. Our results may contribute to the molecular understanding of the pathological filament formation defects of heterozygous DES mutations in cardiomyopathies.

Somatic increase of CCT8 mimics proteostasis of human pluripotent stem cells and extends C. elegans lifespan

Human embryonic stem cells can replicate indefinitely while maintaining their undifferentiated state and, therefore, are immortal in culture. This capacity may demand avoidance of any imbalance in protein homeostasis (proteostasis) that would otherwise compromise stem cell identity. Here we show that human pluripotent stem cells exhibit enhanced assembly of the TRiC/CCT complex, a chaperonin that facilitates the folding of 10% of the proteome. We find that ectopic expression of a single subunit (CCT8) is sufficient to increase TRiC/CCT assembly. Moreover, increased TRiC/CCT complex is required to avoid aggregation of mutant Huntingtin protein. We further show that increased expression of CCT8 in somatic tissues extends Caenorhabditis elegans lifespan in a TRiC/CCT-dependent manner. Ectopic expression of CCT8 also ameliorates the age-associated demise of proteostasis and corrects proteostatic deficiencies in worm models of Huntingtons disease. Our results suggest proteostasis is a common principle that links organismal longevity with hESC immortality.

The novel desmin mutant p.A120D impairs filament formation, prevents intercalated disk localization, and causes sudden cardiac death.

Background—The intermediate filament protein desmin is encoded by the gene DES and contributes to the mechanical stabilization of the striated muscle sarcomere and cell contacts within the cardiac intercalated disk. DES mutations cause severe skeletal and cardiac muscle diseases with heterogeneous phenotypes. Recently, DES mutations were also found in patients with arrhythmogenic right ventricular cardiomyopathy. Currently, the cellular and molecular pathomechanisms of the DES mutations leading to this disease are not exactly known. Methods and Results—We identified the 2 novel variants DES-p.A120D (c.359C>A) and DES-p.H326R (c.977A>G), which were characterized by cell culture experiments and atomic force microscopy. Family analysis indicated a broad spectrum of cardiomyopathies with a striking frequency of arrhythmias and sudden cardiac deaths. The in vitro experiments of desmin-p.A120D reveal a severe intrinsic filament formation defect causing cytoplasmic aggregates in cell lines and of the isolated recombinant protein. Model variants of codon 120 indicated that ionic interactions contribute to this filament formation defect. Ex vivo analysis of ventricular tissue slices revealed a loss of desmin staining within the intercalated disk and severe cytoplasmic aggregate formation, whereas z-band localization was not affected. The functional experiments of desmin-p.H326R did not demonstrate any differences from wild type. Conclusions—Because of the functional in vivo and in vitro characterization, DES-p.A120D has to be regarded as a pathogenic mutation and DES-p.H326R as a rare variant with unknown significance. Presumably, the loss of the desmin-p. A120D filament localization at the intercalated disk explains its clinical arrhythmogenic potential.