Search

Accreditation is one of the approaches to evaluate the quality of health care services, and it’s an essential requirement for the continuity of services. Nursing staff play a major role in the accreditation preparation, process and continuity. The study aimed to assess the nursing perception of the impact of accreditation on the quality of care and safety in a tertiary cardiac care center in the Eastern Province, Kingdome of Saudi Arabia. A cross-sectional descriptive research design was used. A convenient sample of 56 nursing staff was included. The study tool was a self-administered online questionnaire. It consisted of four major sections with 20 subscales rated from 1 to 5 on a Likert scale. Nursing documentation, patient medication information, healthcare-associated infection, leadership and support sections are all included. The study findings indicated that nurses have a 94.6% positive perception of the impact of accreditation on the patient’s safety. This perception was measured in accordance with the items related to international patients’ safety goals. Higher agreement was found on the items of nursing clinical documentation and medication safety process. The study findings support the overall positive impact of accreditation. The study further recommends exploring the nursing perception of accreditation impact in different settings.

Stress granules are membrane-less ribonucleoprotein organelles that assemble upon exposure to stress conditions, but rapidly disassemble upon removal of stress. However, chronic stress can lead to persistent stress granules, a feature of distinct age-related neurodegenerative disorders. Among them, Huntington’s disease (HD), which is caused by mutant expansion of the polyglutamine (polyQ) repeats of huntingtin protein (HTT), leading to its aggregation. To identify modulators of mutant HTT aggregation, we define its interactome in striatal neurons differentiated from patient-derived induced pluripotent stem cells (HD-iPSCs). We find that HTT interacts with G3BP1, a characteristic component of stress granules. Knockdown of G3BP1 increases mutant HTT protein levels and abolishes the ability of iPSCs as well as their differentiated neural counterparts to suppress mutant HTT aggregation. Moreover, loss of G3BP1 hastens polyQ-expanded aggregation and toxicity in the neurons of HD C. elegans models. Likewise, the assembly of G3BP1 into stress granules upon distinct stress conditions also reduces its interaction with HTT in human cells, promoting mutant HTT aggregation. Notably, enhancing the levels of G3BP1 is sufficient to induce proteasomal degradation of mutant HTT and prevent its aggregation, whereas the formation of stress granules blocks these ameliorative effects. In contrast, a mutant G3BP1 variant that cannot accumulate into granules retains its capacity to prevent mutant HTT aggregation even when the cells assemble stress granules. Thus, our findings indicate a direct role of G3BP1 and stress granule assembly in mutant HTT aggregation that may have implications for HD.

Most cardiomyocytes (CMs) in the adult mammalian heart are either binucleated or contain a single polyploid nucleus. Recent studies have shown that polyploidy in CMs plays an important role as an adaptive response to physiological demands and environmental stress and correlates with poor cardiac regenerative ability after injury. However, knowledge about the functional properties of polyploid CMs is limited. In this study, we generated tetraploid pluripotent stem cells (PSCs) by fusion of murine embryonic stem cells (ESCs) and somatic cells isolated from bone marrow or spleen and performed a comparative analysis of the electrophysiological properties of tetraploid fusion-derived PSCs and diploid ESC-derived CMs. Fusion-derived PSCs exhibited characteristics of genuine ESCs and contained a near-tetraploid genome. Ploidy features and marker expression were also retained during the differentiation of fusion-derived cells. Fusion-derived PSCs gave rise to CMs, which were similar to their diploid ESC counterparts in terms of their expression of typical cardiospecific markers, sarcomeric organization, action potential parameters, response to pharmacologic stimulation with various drugs, and expression of functional ion channels. These results suggest that the state of ploidy does not significantly affect the structural and electrophysiological properties of murine PSC-derived CMs. These results extend our knowledge of the functional properties of polyploid CMs and contribute to a better understanding of their biological role in the adult heart.

© The Author(s) 2020., Histones modulate gene expression by chromatin compaction, regulating numerous processes such as differentiation. However, the mechanisms underlying histone degradation remain elusive. Human embryonic stem cells (hESCs) have a unique chromatin architecture characterized by low levels of trimethylated histone H3 at lysine 9 (H3K9me3), a heterochromatin-associated modification. Here we assess the link between the intrinsic epigenetic landscape and ubiquitin-proteasome system of hESCs. We find that hESCs exhibit high expression of the ubiquitin-conjugating enzyme UBE2K. Loss of UBE2K upregulates the trimethyltransferase SETDB1, resulting in H3K9 trimethylation and repression of neurogenic genes during differentiation. Besides H3K9 trimethylation, UBE2K binds histone H3 to induce its polyubiquitination and degradation by the proteasome. Notably, ubc-20, the worm orthologue of UBE2K, also regulates histone H3 levels and H3K9 trimethylation in Caenorhabditis elegans germ cells. Thus, our results indicate that UBE2K crosses evolutionary boundaries to promote histone H3 degradation and reduce H3K9me3 repressive marks in immortal cells., The Deutsche Forschungsgemeinschaft (DFG) (VI742/1-2 and Germany’s Excellence Strategy-CECAD, EXC 2030-390661388), the Else Kröner-Fresenius-Stiftung (2015_A118) and the European Research Council (ERC Starting Grant-677427 StemProteostasis) supported this research. This work was also supported by ERC Consolidator Grant-616499 to T.H., the Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund (POIR.04.04.00-00-5EAB/18-00) to W.P., and the Polish National Science Center (UMO-2016/23/B/NZ3/00753) to A.D. We thank L. Kurian for helpful discussions and comments on the manuscript. We thank the CECAD Proteomics Facility and the Cologne Center for Genomics (CCG) for their contribution and advice in proteomics and RNA sequencing experiments, respectively

We report here a transgenic murine induced pluripotent stem cell (iPSC) line expressing puromycin N-acetyltransferase (PAC) and enhanced green fluorescent protein (EGFP) under the control of α-myosin heavy chain promoter. This transgenic cell line reproducibly differentiates into EGFP-expressing cardiomyocytes (CMs) which can be generated at high purity with puromycin treatment and exhibit molecular and functional properties of immature heart muscle cells. This genetically modified iPSC line can be used for assessment of the utility of CMs for myocardial repair, pharmacological and toxicological applications and development of improved cardiac differentiation protocols.

Summary CAG-repeat expansions in at least eight different genes cause neurodegeneration. The length of the extended polyglutamine stretches in the corresponding proteins is proportionally related to their aggregation propensity. Although these proteins are ubiquitously expressed, they predominantly cause toxicity to neurons. To understand this neuronal hypersensitivity, we generated induced pluripotent stem cell (iPSC) lines of spinocerebellar ataxia type 3 and Huntington’s disease patients. iPSC generation and neuronal differentiation are unaffected by polyglutamine proteins and show no spontaneous aggregate formation. However, upon glutamate treatment, aggregates form in neurons but not in patient-derived neural progenitors. During differentiation, the chaperone network is drastically rewired, including loss of expression of the anti-amyloidogenic chaperone DNAJB6. Upregulation of DNAJB6 in neurons antagonizes glutamate-induced aggregation, while knockdown of DNAJB6 in progenitors results in spontaneous polyglutamine aggregation. Loss of DNAJB6 expression upon differentiation is confirmed in vivo, explaining why stem cells are intrinsically protected against amyloidogenesis and protein aggregates are dominantly present in neurons.

Pluripotent stem cells are invaluable resources to study development and disease, holding a great promise for regenerative medicine. Here we use human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) from patients with Huntington's disease (HD-iPSCs) to shed light into the normal function of huntingtin (HTT) and its demise in disease. We find that HTT binds ATF7IP, a regulator of the histone H3 methyltransferase SETDB1. HTT inhibits the interaction of the ATF7IP-SETDB1 complex with other heterochromatin regulators and transcriptional repressors, maintaining low levels of H3K9 trimethylation (H3K9me3) in hESCs. Loss of HTT promotes global increased H3K9me3 levels and enrichment of H3K9me3 marks at distinct genes, including transcriptional regulators of neuronal differentiation. Although these genes are normally expressed at low amounts in hESCs, HTT knockdown (KD) reduces their induction during neural differentiation. Notably, mutant expanded polyglutamine repeats in HTT diminish its interaction with ATF7IP-SETDB1 complex and trigger H3K9me3 in HD-iPSCs. Conversely, KD of ATF7IP in HD-iPSCs reduces H3K9me3 alterations and ameliorates gene expression changes in their neural counterparts. Taken together, our results indicate ATF7IP as a potential target to correct aberrant H3K9me3 levels induced by mutant HTT.

Huntington's disease (HD) is a fatal neurodegenerative disorder characterized by motor dysfunction, cognitive deficits and psychosis. HD is caused by mutations in the Huntingtin (HTT) gene, resulting in the expansion of polyglutamine (polyQ) repeats in the HTT protein. Mutant HTT is prone to aggregation, and the accumulation of polyQ-expanded fibrils as well as intermediate oligomers formed during the aggregation process contribute to neurodegeneration. Distinct protein homeostasis (proteostasis) nodes such as chaperone-mediated folding and proteolytic systems regulate the aggregation and degradation of HTT. Moreover, polyQ-expanded HTT fibrils and oligomers can lead to a global collapse in neuronal proteostasis, a process that contributes to neurodegeneration. The ability to maintain proteostasis of HTT declines during the aging process. Conversely, mechanisms that preserve proteostasis delay the onset of HD. Here we will review the link between proteostasis, aging and HD-related changes.

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.

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.

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.

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 Ca(2+), Ni(2+), nifedipine, ryanodine, beta-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 Ca(2+) and decreased with nifedipine. Force was reduced by Ni(2+). Isoproterenol (1 microM) increased the force most pronounced in embryonic tissue (207+/-31%, n=7; ESC-CMs: 123+/-5%, n=4; iPS-CMs: 120+/-4%, n=8). EC(50) 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 beta-adrenergic response of iPS-CMs and ESC-CMs.

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.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 beta-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 I(Na), I(f), I(NCX), and I(CaL), respectively, and suggested their involvement in cardiac pacemaking, with I(CaL) being of major importance. Furthermore, iPS-CMs developed and conferred force to avitalized ventricular tissue that was responsive to beta-adrenergic stimulation.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.

We report here the generation of human iPS cell line UKKi009-A from dermal fibroblasts of a patient carrying heterozygous mutation c.3035-3045delTCCCTCGATGC, p.Leu1012Pro (fs*55) in KCNH2 gene leading to long QT syndrome type 2 (LQT2). We used the Sleeping Beauty transposon-based plasmids expressing OSKM along with microRNAs 307/367 to reprogram the fibroblasts. The iPS cells possess pluripotent stem cell characteristics and differentiate to cell lineages of all three germ layers. This cell line can serve as a source for in vitro modeling of LQT2. This cell line is distributed by the European Collection of Authenticated Cell Cultures (ECACC).

Single nucleotide polymorphisms (SNP) refer to single-base differences in DNA sequence between individuals of the same species. In experimental setting, inbred mouse strains can easily be distinguished by their typical SNPs. Therefore, if cell fusion partners are selected to originate from two different genotypes the detection of strain specific SNPs in the genome of fused cells can be utilized as a complimentary method to traditional karyotyping and cell ploidy analyses to monitor the success of the cell fusion procedure and identification of chromosomes from both genotypes in established fusion cell lines. In this chapter, we describe the method for selection and detection of SNPs on each of the 23 pairs of murine chromosome in cell hybrids generated by fusion of murine somatic cells originating from DBA/2J female mice and murine embryonic stem (ES) cells originating from 129/Ola male mice. While parental fusing partners show the presence of only a single strain specific allele the tetraploid fusion hybrid cells harbor alleles originating from both fusing partners indicating that the fusion clones retained both parental nuclei and at least one of each pair of parental autosomes, which were not lost in the course of cell expansion.

Background: Reproducible and efficient differentiation of pluripotent stem cells (PSCs) to cardiomyocytes (CMs) is essential for their use in regenerative medicine, drug testing and disease modeling. The aim of this study was to evaluate the effect of some previously reported cardiogenic substances on cardiac differentiation of mouse PSCs. Methods: Differentiation was performed by embryoid body (EB)-based method using three different murine PSC lines. The differentiation efficiency was monitored by RT-qPCR, immunocytochemistry and flow cytometry, and the effect mechanistically evaluated by transcriptome analysis of treated EBs. Results: Among the five tested compounds (ascorbic acid, dorsomorphin, cyclic adenosine 3',5'-monophosphate, cardiogenol C, cyclosporin A) only ascorbic acid (AA) exerted a strong and reproducible cardiogenic effect in CGR8 cells which was less consistent in other two PSC lines. AA induced only minor changes in transcriptome of CGR8 cells after administration during the initial two days of differentiation. Cardiospecific genes and transcripts involved in angiogenesis, erythropoiesis and hematopoiesis were up-regulated on day 5 but not on days 2 or 3 of differentiation. The cardiac differentiation efficiency was improved when QS11, a small-molecule synergist of Wnt/β-catenin signaling pathway, was added to cultures after AA-treatment. Conclusion: This study demonstrates that only minor transcriptional changes are sufficient for enhancement of cardiogenesis of murine PSCs by AA and that AA and QS11 exhibit synergistic effects and enhance the efficiency of CM differentiation of murine PSCs.

Single nucleotide polymorphisms (SNP) refer to single-base differences in DNA sequence between individuals of the same species. In experimental setting, inbred mouse strains can easily be distinguished by their typical SNPs. Therefore, if cell fusion partners are selected to originate from two different genotypes the detection of strain specific SNPs in the genome of fused cells can be utilized as a complimentary method to traditional karyotyping and cell ploidy analyses to monitor the success of the cell fusion procedure and identification of chromosomes from both genotypes in established fusion cell lines. In this chapter, we describe the method for selection and detection of SNPs on each of the 23 pairs of murine chromosome in cell hybrids generated by fusion of murine somatic cells originating from DBA/2J female mice and murine embryonic stem (ES) cells originating from 129/Ola male mice. While parental fusing partners show the presence of only a single strain specific allele the tetraploid fusion hybrid cells harbor alleles originating from both fusing partners indicating that the fusion clones retained both parental nuclei and at least one of each pair of parental autosomes, which were not lost in the course of cell expansion.

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.

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.

Isolated 4-6 day old RN-CM and HIPSC-CM beat spontaneously under culture conditions and appear to express all the ionic channels associated with the cardiac myocyte phenotype. Both Cell types continued spontaneous beating and Ca2+ oscillations at −60mV holding potentials where Na+, K+, If, and Ca2 currents were not allowed to activate. Nevertheless, inward transients of NCX currents (INCX) occurred regularly (50-150 beats per minute at ∼25 oC) with spontaneous releases of Ca2+, using Cs+-based dialyzing solutions containing 0.1-0.2 mM concentrations of Fura 2 or EGTA. Caffeine-induced Ca2+-releases also activated large INCX in both RN-CM (1.7 pA/pF, n=13) and HIPSC-CM (3.9 pA/pF, n=9) that were 2-4 times larger than those of mature rat or human myocytes. The rate and magnitude of Ca2+-oscillations and INCX increased on adrenergic stimulation, and rapid increases of [Ca2+]o from 2-5 mM. Withdrawal of [Ca2+]o and application of NCX-blocker (KBR-7943) or tetracaine also rapidly and reversibly inhibited spontaneous Ca2+-oscillations. Xestospongine C, and 2-APB applications, to probe the role IP3-signaling failed to alter spontaneous beating, as did Ca2+-channel blocker (nifedipine) and NO-synthase inhibitor L-NAME. Surprisingly, application of low concentrations of mitochondrial uncouplers (5-50 nM FCCP or 100-200 μM DNP) suppressed the spontaneous Ca2+-oscillations rapidly and reversibly in both cell types and inhibited the rate of uptake of caffeine-induced release of Ca2+. Blockers of mitochondrial-uniporter, or mitochondrial NCX were ineffective in modulating the frequency of spontaneous beating. Our data suggests that mechanisms of spontaneous pacing are similar in HIPSC-CM and RN-CM, and are mediated by possible Ca2+cross-talk between NCX, RyR/SR, and mitochondria. It is as yet unclear whether the mitochondrial Ca2+ cycling primarily initiates or modulates the spontaneous Ca2+-oscillations and beating. (NIH HL16152 and HL 107600).

iPSCS were created from skin biopsies of control subjects and a patient with catecholaminergic polymorphic ventricular tachycardia associated with point mutation (F2483I) in the FKBP12.6 binding domain of RYR2. Here we used rapid 2-dimensional confocal fluorescence imaging in patch-clamped cells to investigate Ca2+ signaling in hIPSCs in which cardiac phenotype was indicated by spontaneous beating, ICa-gated Ca2+ release, and protein expression. hIPSC shapes varied from spherical to elongated with a sarcomeric pattern. The ICa-gated Ca2+ release in patient-derived IPSCs occurred first at the periphery of the cells, but unlike control cells, continued even after deactivation of ICa. Exposures to cAMP and forskolin suppressed ICa-gated Ca2+ release, even though caffeine-induced release showed that SR Ca2+ stores were intact. In elongated cells, ICa-gated Ca2+ release caused brief localized Ca2+ releases (sparks) in a sarcomeric pattern, somewhat similar to mature ventricular cardiomyocytes, but sparking activity continued long after repolarization, consistent with over-active RyRs. In some round-shaped cells, clamped at −60 mV, spontaneous Ca2+ release activity, of variable frequency and loci, were frequently observed. In other voltage clamped cells we found propagated Ca2+ waves of random distribution and dispersion. Our results suggest that hIPSC-derived cardiomyocytes produce cardiac-type Ica-gated Ca2+-signaling in control and in patient-derived cells, but the mutant hIPSCs show Ca2+-signaling phenotype consistent with over-active RyRs and the pathology of the CPVT disease. (Supported by NIH HL16152 and HL 107600).

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 RT-PCR, immunocytochemistry, and electrophysiology. As key markers of cardiac lineages, transcripts for Nkx2.5, alphaMHC, 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.

Purpose: Induced pluripotent stem cell derived cardiomyocytes (iPS-CM) are suitable for cardiac cell replacement therapy since they can integrate into host myocardium after transplantation and thereby improve heart function. However, highly purification is needed to avoid teratogenic risk but it leads to poor persistence of transplanted iPS-CM which we here tried to improve by co-transplantation of non-cardiomyocytes (non-CM). Methods: Male iPS-CM were derived from transgenic murine induced pluripotent stem cells and highly purified using an antibiotic resistance under a cardiac specific promoter. Intramyocardial injection of 300,000 iPS-CM with or without 300,000 male wild-type murine non-CM (embryonic fibroblasts [+MEF] or adult mesenchymal bone marrow cells [+MSC]) was performed into healthy hearts of syngeneic female wild-type mice. Immediately (0h) or after 24h, 48h or 7 days (each n≥4 per group), hearts were harvested and the number of persisting transplanted cells was determined by quantitative real-time PCR with specific primer for SRY or transgene. One additional cell aliquot was mixed with an explanted native heart ex vivo as control (=100%) for every surgery day. Results: Immediately after transplantation of iPS-CM alone (0h), 28.4±4.0% of transplanted cells were detected in recipient hearts and the number decreased to 2.6±0.9% at 6h (p