Publications by authors named "Kaomei Guan"

65 Publications

A calcineurin-mediated scaling mechanism that controls a K-leak channel to regulate morphogen and growth factor transcription.

Elife 2021 Apr 8;10. Epub 2021 Apr 8.

School of Life Sciences and Technology, ShanghaiTech University, Shanghai, China.

The increase in activity of the two-pore potassium-leak channel Kcnk5b maintains allometric juvenile growth of adult zebrafish appendages. However, it remains unknown how this channel maintains allometric growth and how its bioelectric activity is regulated to scale these anatomical structures. We show the activation of Kcnk5b is sufficient to activate several genes that are part of important development programs. We provide transplantation evidence that the activation of gene transcription is cell autonomous. We also show that Kcnk5b will induce the expression of different subsets of the tested developmental genes in different cultured mammalian cell lines, which may explain how one electrophysiological stimulus can coordinately regulate the allometric growth of diverse populations of cells in the fin that use different developmental signals. We also provide evidence that the post-translational modification of serine 345 in Kcnk5b by calcineurin regulates channel activity to scale the fin. Thus, we show how an endogenous bioelectric mechanism can be regulated to promote coordinated developmental signaling to generate and scale a vertebrate appendage.
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http://dx.doi.org/10.7554/eLife.60691DOI Listing
April 2021

Generation of iPSC lines from CPVT patient carrying heterozygous mutation p.A2254V in the ryanodine receptor 2 gene.

Stem Cell Res 2021 Feb 19;53:102259. Epub 2021 Feb 19.

Institute of Pharmacology and Toxicology, Technische Universität Dresden, Germany; Clinic for Cardiology and Pneumology, Universitätsmedizin Göttingen, Germany. Electronic address:

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a severe inheritable cardiac disorder, which is characterized by life-threatening cardiac arrhythmias, syncope, seizures, or sudden cardiac death in response to physical exercise or emotional stress. This inherited disease is predominantly caused by mutations in the ryanodine receptor type 2 (RYR2). To minimize the cell line variations for disease modeling, we generated two induced pluripotency stem cell lines (hiPSCs: isCPVT1-2 and isCPVT1-3) from skin fibroblasts of one CPVT patient carrying the p.A2254V mutation using CytoTune2.0 Sendai virus cocktail for non-integration reprogramming. All generated iPSCs maintained pluripotency, normal karyotype, and spontaneous in vivo and in vitro differentiation capacity.
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http://dx.doi.org/10.1016/j.scr.2021.102259DOI Listing
February 2021

Electrophysiological Abnormalities in VLCAD Deficient hiPSC-Cardiomyocytes Do not Improve with Carnitine Supplementation.

Front Pharmacol 2020 12;11:616834. Epub 2021 Jan 12.

Laboratory Genetic Metabolic Diseases, Amsterdam UMC, University of Amsterdam, Amsterdam Gastroenterology and Metabolism, Amsterdam Cardiovascular Sciences, Amsterdam, Netherlands.

Patients with a deficiency in very long-chain acyl-CoA dehydrogenase (VLCAD), an enzyme that is involved in the mitochondrial beta-oxidation of long-chain fatty acids, are at risk for developing cardiac arrhythmias. In human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs), VLCAD deficiency (VLCADD) results in a series of abnormalities, including: 1) accumulation of long-chain acylcarnitines, 2) action potential shortening, 3) higher systolic and diastolic intracellular Ca concentrations, and 4) development of delayed afterdepolarizations. In the fatty acid oxidation process, carnitine is required for bidirectional transport of acyl groups across the mitochondrial membrane. Supplementation has been suggested as potential therapeutic approach in VLCADD, but its benefits are debated. Here, we studied the effects of carnitine supplementation on the long-chain acylcarnitine levels and performed electrophysiological analyses in VLCADD patient-derived hiPSC-CMs with a gene mutation (p.Val283Ala/p.Glu381del). Under standard culture conditions, VLCADD hiPSC-CMs showed high concentrations of long-chain acylcarnitines, short action potentials, and high delayed afterdepolarizations occurrence. Incubation of the hiPSC-CMs with 400 µM L-carnitine for 48 h led to increased long-chain acylcarnitine levels both in medium and cells. In addition, carnitine supplementation neither restored abnormal action potential parameters nor the increased occurrence of delayed afterdepolarizations in VLCADD hiPSC-CMs. We conclude that long-chain acylcarnitine accumulation and electrophysiological abnormalities in VLCADD hiPSC-CMs are not normalized by carnitine supplementation, indicating that this treatment is unlikely to be beneficial against cardiac arrhythmias in VLCADD patients.
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http://dx.doi.org/10.3389/fphar.2020.616834DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7883678PMC
January 2021

Disease Phenotypes and Mechanisms of iPSC-Derived Cardiomyocytes From Brugada Syndrome Patients With a Loss-of-Function SCN5A Mutation.

Front Cell Dev Biol 2020 22;8:592893. Epub 2020 Oct 22.

Institute of Pharmacology and Toxicology, Technische Universität Dresden, Dresden, Germany.

Brugada syndrome (BrS) is one of the major causes of sudden cardiac death in young people, while the underlying mechanisms are not completely understood. Here, we investigated the pathophysiological phenotypes and mechanisms using induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) from two BrS patients (BrS-CMs) carrying a heterozygous mutation p.S1812X. Compared to CMs derived from healthy controls (Ctrl-CMs), BrS-CMs displayed a 50% reduction of density, a 69.5% reduction of Na1.5 expression, and the impaired localization of Na1.5 and connexin 43 (Cx43) at the cell surface. BrS-CMs exhibited reduced action potential (AP) upstroke velocity and conduction slowing. The in BrS-CMs was significantly augmented, and the window current probability was increased. Our data indicate that the electrophysiological mechanisms underlying arrhythmia in BrS-CMs may involve both depolarization and repolarization disorders. Cilostazol and milrinone showed dramatic inhibitions of in BrS-CMs and alleviated the arrhythmic activity, suggesting their therapeutic potential for BrS patients.
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http://dx.doi.org/10.3389/fcell.2020.592893DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642519PMC
October 2020

Repurposing Antidiabetic Drugs for Cardiovascular Disease.

Front Physiol 2020 15;11:568632. Epub 2020 Sep 15.

Institute of Pharmacology and Toxicology, Technische Universität Dresden, Dresden, Germany.

Metabolic diseases and diabetes represent an increasing global challenge for human health care. As associated with a strongly elevated risk of developing atherosclerosis, kidney failure and death from myocardial infarction or stroke, the treatment of diabetes requires a more effective approach than lowering blood glucose levels. This review summarizes the evidence for the cardioprotective benefits induced by antidiabetic agents, including sodium-glucose cotransporter 2 inhibitor (SGLT2i) and glucagon-like peptide-1 receptor agonist (GLP1-RA), along with sometimes conversely discussed effects of dipeptidyl peptidase-4 inhibitor (DPP4i) and metformin in patients with high cardiovascular risk with or without type 2 diabetes. Moreover, the proposed mechanisms of the different drugs are described based on the results of preclinical studies. Recent cardiovascular outcome trials unexpectedly confirmed a beneficial effect of GLP-1RA and SGLT2i in type 2 diabetes patients with high cardiovascular risk and with standard care, which was independent of glycaemic control. These results triggered a plethora of studies to clarify the underlying mechanisms and the relevance of these effects. Taken together, the available data strongly highlight the potential of repurposing the original antidiabetics GLP1-RA and SGLT2i to improve cardiovascular outcome even in non-diabetic patients with cardiovascular diseases.
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http://dx.doi.org/10.3389/fphys.2020.568632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7522553PMC
September 2020

IP3R-Mediated Compensatory Mechanism for Calcium Handling in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes With Cardiac Ryanodine Receptor Deficiency.

Front Cell Dev Biol 2020 12;8:772. Epub 2020 Aug 12.

Institute of Pharmacology and Toxicology, Technische Universität Dresden, Dresden, Germany.

In adult cardiomyocytes (CMs), the type 2 ryanodine receptor (RYR2) is an indispensable Ca release channel that ensures the integrity of excitation-contraction coupling, which is fundamental for every heartbeat. However, the role and importance of RYR2 during human embryonic cardiac development are still poorly understood. Here, we generated two human induced pluripotent stem cell (iPSC)-based knockout (RYR2) lines using the CRISPR/Cas9 gene editing technology. We found that RYR2-iPSCs could differentiate into CMs with the efficiency similar to control-iPSCs (Ctrl-iPSCs); however, the survival of iPSC-CMs was markedly affected by the lack of functional RYR2. While Ctrl-iPSC-CMs exhibited regular Ca handling, we observed significantly reduced frequency and intense abnormalities of Ca transients in RYR2-iPSC-CMs. Ctrl-iPSC-CMs displayed sensitivity to extracellular Ca ([Ca ]) and caffeine in a concentration-dependent manner, while RYR2-iPSC-CMs showed inconsistent reactions to [Ca ] and were insensitive to caffeine, indicating there is no RYR2-mediated Ca release from the sarcoplasmic reticulum (SR). Instead, compensatory mechanism for calcium handling in RYR2-iPSC-CMs is partially mediated by the inositol 1,4,5-trisphosphate receptor (IP3R). Similar to Ctrl-iPSC-CMs, SR Ca refilling in RYR2-iPSC-CMs is mediated by SERCA. Additionally, RYR2-iPSC-CMs showed a decreased beating rate and a reduced peak amplitude of L-type Ca current. These findings demonstrate that RYR2 is not required for CM lineage commitment but is important for CM survival and contractile function. IP3R-mediated Ca release is one of the major compensatory mechanisms for Ca cycling in human CMs with the RYR2 deficiency.
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http://dx.doi.org/10.3389/fcell.2020.00772DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7434870PMC
August 2020

Role of Leptin in Cardiovascular Diseases.

Front Endocrinol (Lausanne) 2020 16;11:354. Epub 2020 Jun 16.

Medical Faculty Carl Gustav Carus, Institute of Pharmacology and Toxicology, Technische Universität Dresden, Dresden, Germany.

The adipocyte-derived adipokine leptin exerts pleiotropic effects, which are essential for the regulation of energy balance and cell metabolism, for controlling inflammatory and immune responses, and for the maintenance of homeostasis of the cardiovascular system. Leptin resistance in obese or type 2 diabetes mellitus (T2DM) patients is defined as a decrease in tissue response to leptin. In the cardiovascular system, leptin resistance exhibits the adverse effect on the heart's response to stress conditions and promoting cardiac remodeling due to impaired cardiac metabolism, increased fibrosis, vascular dysfunction, and enhanced inflammation. Leptin resistance or leptin signaling deficiency results in the risk increase of cardiac dysfunction and heart failure, which is a leading cause of obesity- and T2DM-related morbidity and mortality. Animal studies using leptin- and leptin receptor- (Lepr) deficient rodents have provided many useful insights into the underlying molecular and pathophysiological mechanisms of obese- and T2DM-associated metabolic and cardiovascular diseases. However, none of the animal models used so far can fully recapitulate the phenotypes of patients with obese or T2DM. Therefore, the role of leptin in the human cardiovascular system, and whether leptin affects cardiac function directly or acts through a leptin-regulated neurohumoral pathway, remain elusive. As the prevalence of obesity and diabetes is continuously increasing, strategies are needed to develop and apply human cell-based models to better understand the precise role of leptin directly in different cardiac cell types and to overcome the existing translational barriers. The purpose of this review is to discuss the mechanisms associated with leptin signaling deficiency or leptin resistance in the development of metabolic and cardiovascular diseases. We analyzed and comprehensively addressed substantial findings in pathophysiological mechanisms in commonly used leptin- or Lepr-deficient rodent models and highlighted the differences between rodents and humans. This may open up new strategies to develop directly and reliably applicable models, which resemble the human pathophysiology in order to advance health care management of obesity- and T2DM-related cardiovascular complications.
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http://dx.doi.org/10.3389/fendo.2020.00354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7325922PMC
June 2020

Induced pluripotent stem cells of patients with Tetralogy of Fallot reveal transcriptional alterations in cardiomyocyte differentiation.

Sci Rep 2020 07 2;10(1):10921. Epub 2020 Jul 2.

Cardiovascular Genetics, Charité - Universitätsmedizin Berlin, Berlin, Germany.

Patient-specific induced pluripotent stem cells (ps-iPSCs) and their differentiated cell types are a powerful model system to gain insight into mechanisms driving early developmental and disease-associated regulatory networks. In this study, we use ps-iPSCs to gain insights into Tetralogy of Fallot (TOF), which represents the most common cyanotic heart defect in humans. iPSCs were generated and further differentiated to cardiomyocytes (CMs) using standard methods from two well-characterized TOF patients and their healthy relatives serving as controls. Patient-specific expression patterns and genetic variability were investigated using whole genome and transcriptome sequencing data. We first studied the clonal mutational burden of the derived iPSCs. In two out of three iPSC lines of patient TOF-01, we found a somatic mutation in the DNA-binding domain of tumor suppressor P53, which was not observed in the genomic DNA from blood. Further characterization of this mutation showed its functional impact. For patient TOF-02, potential disease-relevant differential gene expression between and across cardiac differentiation was shown. Here, clear differences at the later stages of differentiation could be observed between CMs of the patient and its controls. Overall, this study provides first insights into the complex molecular mechanisms underlying iPSC-derived cardiomyocyte differentiation and its transcriptional alterations in TOF.
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http://dx.doi.org/10.1038/s41598-020-67872-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7331606PMC
July 2020

Electrophysiological Abnormalities in VLCAD Deficient hiPSC-Cardiomyocytes Can Be Improved by Lowering Accumulation of Fatty Acid Oxidation Intermediates.

Int J Mol Sci 2020 Apr 8;21(7). Epub 2020 Apr 8.

Department of Clinical and Experimental Cardiology, Heart Center, Amsterdam Cardiovascular Sciences, Amsterdam UMC, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands.

Patients with very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) can present with life-threatening cardiac arrhythmias. The pathophysiological mechanism is unknown. We reprogrammed fibroblasts from one mildly and one severely affected VLCADD patient, into human induced pluripotent stem cells (hiPSCs) and differentiated these into cardiomyocytes (VLCADD-CMs). VLCADD-CMs displayed shorter action potentials (APs), more delayed afterdepolarizations (DADs) and higher systolic and diastolic intracellular Ca concentration ([Ca]) than control CMs. The mitochondrial booster resveratrol mitigated the biochemical, electrophysiological and [Ca] changes in the mild but not in the severe VLCADD-CMs. Accumulation of potentially toxic intermediates of fatty acid oxidation was blocked by substrate reduction with etomoxir. Incubation with etomoxir led to marked prolongation of AP duration and reduced DADs and [Ca] in both VLCADD-CMs. These results provide compelling evidence that reduced accumulation of fatty acid oxidation intermediates, either by enhanced fatty acid oxidation flux through increased mitochondria biogenesis (resveratrol) or by inhibition of fatty acid transport into the mitochondria (etomoxir), rescues pro-arrhythmia defects in VLCADD-CMs and open doors for new treatments.
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http://dx.doi.org/10.3390/ijms21072589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7177397PMC
April 2020

Marine biomaterials: Biomimetic and pharmacological potential of cultivated Aplysina aerophoba marine demosponge.

Mater Sci Eng C Mater Biol Appl 2020 Apr 17;109:110566. Epub 2019 Dec 17.

Institute of Electronics and Sensor Materials, Technische Universität Bergakademie Freiberg, Freiberg 09599, Germany. Electronic address:

Marine demosponges of the Verongiida order are considered a gold-mine for bioinspired materials science and marine pharmacology. The aim of this work was to simultaneously isolate selected bromotyrosines and unique chitinous structures from A. aerophoba and to propose these molecules and biomaterials for possible application as antibacterial and antitumor compounds and as ready-to-use scaffolds for cultivation of cardiomyocytes, respectively. Among the extracted bromotyrosines, the attention has been focused on aeroplysinin-1 that showed interesting unexpected growth inhibition properties for some Gram-negative clinical multi-resistant bacterial strains, such as A. baumannii and K. pneumoniae, and on aeroplysinin-1 and on isofistularin-3 for their anti-tumorigenic activity. For both compounds, the effects are cell line dependent, with significant growth inhibition activity on the neuroblastoma cell line SH-SY5Y by aeroplysinin-1 and on breast cancer cell line MCF-7 by isofistularin-3. In this study, we also compared the cultivation of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) on the A. aerophoba chitinous scaffolds, in comparison to chitin structures that were pre-coated with Geltrex™, an extracellular matrix mimetic which is used to enhance iPSC-CM adhesion. The iPSC-CMs on uncoated and pure chitin structures started contracting 24 h after seeding, with comparable behaviour observed on Geltrex-coated cell culture plates, confirming the biocompatibility of the sponge biomaterial with this cell type. The advantage of A. aerophoba is that this source organism does not need to be collected in large quantities to supply the necessary amount for further pre-clinical studies before chemical synthesis of the active compounds will be available. A preliminary analysis of marine sponge bioeconomy as a perspective direction for application of biomaterials and secondary bioactive metabolites has been finally performed for the first time.
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http://dx.doi.org/10.1016/j.msec.2019.110566DOI Listing
April 2020

Targeting alleviates myocardial insulin resistance and prevents ischemia-induced heart failure.

Elife 2020 03 30;9. Epub 2020 Mar 30.

Faculty of Biology, Medicine, and Health, the University of Manchester, Manchester, United Kingdom.

Myocardial insulin resistance contributes to heart failure in response to pathological stresses, therefore, a therapeutic strategy to maintain cardiac insulin pathways requires further investigation. We demonstrated that insulin receptor substrate 1 (IRS1) was reduced in failing mouse hearts post-myocardial infarction (MI) and failing human hearts. The mice manifesting severe cardiac dysfunction post-MI displayed elevated in the myocardium. Ischemia-upregulated promoted degradation. Using rat cardiomyocytes and human-induced pluripotent stem cell-derived cardiomyocytes, we elucidated that mitogen-activated protein kinase 7 (MAPK7, also known as ERK5)-mediated CCAAT/enhancer-binding protein beta (CEBPβ) transcriptionally represses under hypoxia. Therapeutically, functional studies demonstrated gene therapy-delivered cardiac-specific MAPK7 restoration or overexpression of CEBPβ impeded cardiac injury after MI, at least partly due to normalization of . Furthermore, inhibition of preserved and ameliorated cardiac dysfunction post-MI. In conclusion, we reveal that targeting mitigates myocardial insulin resistance, thereafter slowing down the progression of heart failure post-ischemia.
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http://dx.doi.org/10.7554/eLife.54298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7124275PMC
March 2020

Absence of Functional Na1.8 Channels in Non-diseased Atrial and Ventricular Cardiomyocytes.

Cardiovasc Drugs Ther 2019 12;33(6):649-660

Department of Experimental Cardiology, Amsterdam UMC, Meibergdreef 15, Amsterdam, 1105 AZ, The Netherlands.

Purpose: Several studies have indicated a potential role for SCN10A/Na1.8 in modulating cardiac electrophysiology and arrhythmia susceptibility. However, by which mechanism SCN10A/Na1.8 impacts on cardiac electrical function is still a matter of debate. To address this, we here investigated the functional relevance of Na1.8 in atrial and ventricular cardiomyocytes (CMs), focusing on the contribution of Na1.8 to the peak and late sodium current (I) under normal conditions in different species.

Methods: The effects of the Na1.8 blocker A-803467 were investigated through patch-clamp analysis in freshly isolated rabbit left ventricular CMs, human left atrial CMs and human-induced pluripotent stem cell-derived CMs (hiPSC-CMs).

Results: A-803467 treatment caused a slight shortening of the action potential duration (APD) in rabbit CMs and hiPSC-CMs, while it had no effect on APD in human atrial cells. Resting membrane potential, action potential (AP) amplitude, and AP upstroke velocity were unaffected by A-803467 application. Similarly, I density was unchanged after exposure to A-803467 and Na1.8-based late I was undetectable in all cell types analysed. Finally, low to absent expression levels of SCN10A were observed in human atrial tissue, rabbit ventricular tissue and hiPSC-CMs.

Conclusion: We here demonstrate the absence of functional Na1.8 channels in non-diseased atrial and ventricular CMs. Hence, the association of SCN10A variants with cardiac electrophysiology observed in, e.g. genome wide association studies, is likely the result of indirect effects on SCN5A expression and/or Na1.8 activity in cell types other than CMs.
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http://dx.doi.org/10.1007/s10557-019-06925-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994555PMC
December 2019

Establishment of an automated patch-clamp platform for electrophysiological and pharmacological evaluation of hiPSC-CMs.

Stem Cell Res 2019 12 18;41:101662. Epub 2019 Nov 18.

Institute of Pharmacology and Toxicology, Technische Universität Dresden, Fetscherstraße 74, 01307 Dresden, Germany. Electronic address:

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have evolved into widely used and reliable cell sources for modeling cardiovascular channelopathies and for drug safety pharmacology. However, the electrophysiological and pharmacological applications of hiPSC-CMs are hampered by manual patch-clamp technique, which is labor-intensive and generates low output. The automated patch-clamp technique is showing potential to overcome this problem. Here, we describe a new dissociation method, with which we can harvest a vast number of single relaxed hiPSC-CMs with smooth membrane suited for automated patch-clamp. Using the automated whole-cell patch-clamp technology, we report a high success rate for cell capture and whole-cell access (around 70%). We are able to identify and record several currents and paced action potentials (APs) with different success rates, including Na current (I), L-type Ca current (I), two specific K currents, the transient outward K current (I) and the inward rectifier K current (I). Moreover, we successfully applied dynamic current-clamp to virtually increase I for AP recordings. Our study suggests that automated patch-clamp technology could be used to investigate the relevant ionic currents and APs in hiPSC-CMs. The combination of automated patch-clamp and hiPSC-CM technologies promises a wide range of applications in the future.
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http://dx.doi.org/10.1016/j.scr.2019.101662DOI Listing
December 2019

Enhanced structural maturation of human induced pluripotent stem cell-derived cardiomyocytes under a controlled microenvironment in a microfluidic system.

Acta Biomater 2020 01 26;102:273-286. Epub 2019 Nov 26.

Institute of Pharmacology and Toxicology, Technische Universität Dresden, Fetscherstr. 74, 01307 Dresden, Germany. Electronic address:

The lack of a fully developed human cardiac model in vitro hampers the progress of many biomedical research fields including pharmacology, developmental biology, and disease modeling. Currently, available methods may only differentiate human induced pluripotent stem cells (iPSCs) into immature cardiomyocytes. To achieve cardiomyocyte maturation, appropriate modulation of cellular microenvironment is needed. This study aims to optimize a microfluidic system that enhances maturation of human iPSC-derived cardiomyocytes (iPSC-CMs) through cyclic pulsatile hemodynamic forces. Human iPSC-CMs cultured in the microfluidic system show increased alignment and contractility and appear more rod-like shaped with increased cell size and increased sarcomere length when compared to static cultures. Increased complexity and density of the mitochondrial network in iPSC-CMs cultured in the microfluidic system are in line with expression of mitochondrial marker genes MT-CO1 and OPA1. Moreover, the optimized microfluidic system is capable of stably maintaining controlled oxygen levels and inducing hypoxia, revealed by increased expression of HIF1α and EGLN2 as well as changes in contraction parameters in iPSC-CMs. In summary, this microfluidic system boosts the structural maturation of iPSC-CM culture and could serve as an advanced in vitro cardiac model for biomedical research in the future. STATEMENT OF SIGNIFICANCE: The availability of in vitro human cardiomyocytes generated from induced pluripotent stem cells (iPSCs) opens the possibility to develop human in vitro heart models for disease modeling and drug testing. However, iPSC-derived cardiomyocytes remain structurally and functionally immature, which hinders their application. In this manuscript, we present an optimized and complete microfluidic system that enhances maturation of iPSC-derived cardiomyocytes through physiological cyclic pulsatile hemodynamic forces. Furthermore, we improved our microfluidic system by using a closed microfluidic recirculation and oxygen exchangers to achieve and maintain low oxygen in the culture chambers, which is suitable for mimicking the hypoxic condition and studying the pathophysiological mechanisms of human diseases in vitro. In the future, a variety of technologies including 3D tissue engineering could be integrated into our system, which may greatly extend the use of iPSC-derived cardiac models in drug development and disease modeling.
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http://dx.doi.org/10.1016/j.actbio.2019.11.044DOI Listing
January 2020

Naturally Drug-Loaded Chitin: Isolation and Applications.

Mar Drugs 2019 Oct 10;17(10). Epub 2019 Oct 10.

Institute of Electronic and Sensor Materials, TU Bergakademie Freiberg, Gustav-Zeuner Str. 3, Freiberg 09599, Germany.

Naturally occurring three-dimensional (3D) biopolymer-based matrices that can be used in different biomedical applications are sustainable alternatives to various artificial 3D materials. For this purpose, chitin-based structures from marine sponges are very promising substitutes. Marine sponges from the order Verongiida (class Demospongiae) are typical examples of demosponges with well-developed chitinous skeletons. In particular, species belonging to the family Ianthellidae possess chitinous, flat, fan-like fibrous skeletons with a unique, microporous 3D architecture that makes them particularly interesting for applications. In this work, we focus our attention on the demosponge (Linnaeus, 1759) for simultaneous extraction of both naturally occurring ("ready-to-use") chitin scaffolds, and biologically active bromotyrosines which are recognized as potential antibiotic, antitumor, and marine antifouling substances. We show that selected bromotyrosines are located within pigmental cells which, however, are localized within chitinous skeletal fibers of . A two-step reaction provides two products: treatment with methanol extracts the bromotyrosine compounds bastadin 25 and araplysillin-I N20 sulfamate, and a subsequent treatment with acetic acid and sodium hydroxide exposes the 3D chitinous scaffold. This scaffold is a mesh-like structure, which retains its capillary network, and its use as a potential drug delivery biomaterial was examined for the first time. The results demonstrate that sponge-derived chitin scaffolds, impregnated with decamethoxine, effectively inhibit growth of the human pathogen in an agar diffusion assay.
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http://dx.doi.org/10.3390/md17100574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6835269PMC
October 2019

Spider Chitin: An Ultrafast Microwave-Assisted Method for Chitin Isolation from Spider Molt Cuticle.

Molecules 2019 Oct 16;24(20). Epub 2019 Oct 16.

Institute of Electronics and Sensor Materials, TU Bergakademie Freiberg, 09599 Freiberg, Germany.

Chitin, as a fundamental polysaccharide in invertebrate skeletons, continues to be actively investigated, especially with respect to new sources and the development of effective methods for its extraction. Recent attention has been focused on marine crustaceans and sponges; however, the potential of spiders (order Araneae) as an alternative source of tubular chitin has been overlooked. In this work, we focused our attention on chitin from up to 12 cm-large Theraphosidae spiders, popularly known as tarantulas or bird-eating spiders. These organisms "lose" large quantities of cuticles during their molting cycle. Here, we present for the first time a highly effective method for the isolation of chitin from spider molt cuticle, as well as its identification and characterization using modern analytical methods. We suggest that the tube-like molt cuticle of this spider can serve as a naturally prefabricated and renewable source of tubular chitin with high potential for application in technology and biomedicine.
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http://dx.doi.org/10.3390/molecules24203736DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6833065PMC
October 2019

Naturally Prefabricated Marine Biomaterials: Isolation and Applications of Flat Chitinous 3D Scaffolds from (Demospongiae: Verongiida).

Int J Mol Sci 2019 Oct 15;20(20). Epub 2019 Oct 15.

Institute of Electronics and Sensor Materials, TU Bergakademie Freiberg, Gustav-Zeuner str. 3, 09599 Freiberg, Germany.

Marine sponges remain representative of a unique source of renewable biological materials. The demosponges of the family Ianthellidae possess chitin-based skeletons with high biomimetic potential. These three-dimensional (3D) constructs can potentially be used in tissue engineering and regenerative medicine. In this study, we focus our attention, for the first time, on the marine sponge Bergquist & Kelly-Borges, 1995 (Demospongiae: Verongida: Ianthellidae) as a novel potential source of naturally prestructured bandage-like 3D scaffolds which can be isolated simultaneously with biologically active bromotyrosines. Specifically, translucent and elastic flat chitinous scaffolds have been obtained after bromotyrosine extraction and chemical treatments of the sponge skeleton with alternate alkaline and acidic solutions. For the first time, cardiomyocytes differentiated from human induced pluripotent stem cells (iPSC-CMs) have been used to test the suitability of chitinous skeleton as ready-to-use scaffold for their cell culture. Results reveal a comparable attachment and growth on isolated chitin-skeleton, compared to scaffolds coated with extracellular matrix mimetic Geltrex. Thus, the natural, unmodified cleaned sponge skeleton can be used to culture iPSC-CMs and 3D tissue engineering. In addition, chitin-based scaffolds demonstrate strong and efficient capability to absorb blood deep into the microtubes due to their excellent capillary effect. These findings are suggestive of the future development of new sponge chitin-based absorbable hemostats as alternatives to already well recognized cellulose-based fabrics.
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http://dx.doi.org/10.3390/ijms20205105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6829448PMC
October 2019

Spider Chitin. The biomimetic potential and applications of Caribena versicolor tubular chitin.

Carbohydr Polym 2019 Dec 8;226:115301. Epub 2019 Sep 8.

Institute of Electronics and Sensor Materials, TU Bergakademie Freiberg, Freiberg 09599, Germany. Electronic address:

Diverse fields of modern technology and biomedicine can benefit from the application of ready-to-use chitin-based scaffolds. In this work we show for the first time the applicability of tubular and porous chitin from Caribena versicolor spiders as a scaffold for the development of an effective CuO/Cu(OH) catalyst for the reduction of 4-nitrophenol (4-NP) to 4-aminophenol (4-AM), and as a scaffold for the tissue engineering of selected cells. The formation of CuO/Cu(OH) phases on and within the chitinous tubes leads to a hybrid material with excellent catalytic performance with respect to the reduction of p-nitrophenol. On the other hand, experimental results provide for the first time strong evidence for the biocompatibility of spider chitin with different cell types, a human progenitor cell line (hPheo1), as well as cardiomyocytes differentiated from induced pluripotent stem cells (iPSC-CMs) that were cultured on a tube-like scaffold.
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http://dx.doi.org/10.1016/j.carbpol.2019.115301DOI Listing
December 2019

Control of p21Cip by BRCA1-associated protein is critical for cardiomyocyte cell cycle progression and survival.

Cardiovasc Res 2020 03;116(3):592-604

Department of Cardiology and Pulmonology, Georg-August University, Robert-Koch Str. 40, 37075 Göttingen, Germany.

Aims: Identifying the key components in cardiomyocyte cell cycle regulation is of relevance for the understanding of cardiac development and adaptive and maladaptive processes in the adult myocardium. BRCA1-associated protein (BRAP) has been suggested as a cytoplasmic retention factor for several proteins including Cyclin-dependent-kinase inhibitor p21Cip. We observed profound expressional changes of BRAP in early postnatal myocardium and investigated the impact of BRAP on cardiomyocyte cell cycle regulation.

Methods And Results: General knockout of Brap in mice evoked embryonic lethality associated with reduced myocardial wall thickness and lethal cardiac congestion suggesting a prominent role for BRAP in cardiomyocyte proliferation. αMHC-Cre driven cardiomyocyte-specific knockout of Brap also evoked lethal cardiac failure shortly after birth. Likewise, conditional cardiomyocyte-specific Brap deletion using tamoxifen-induced knockout in adult mice resulted in marked ventricular dilatation and heart failure 3 weeks after induction. Several lines of evidence suggest that Brap deletion evoked marked inhibition of DNA synthesis and cell cycle progression. In cardiomyocytes with proliferative capacity, this causes developmental arrest, whereas in adult hearts loss of BRAP-induced apoptosis. This is explained by altered signalling through p21Cip which we identify as the link between BRAP and cell cycle/apoptosis. BRAP deletion enhanced p21Cip expression, while BRAP overexpression in cardiomyocyte-specific transgenic mice impeded p21Cip expression. That was paralleled by enhanced nuclear Ki-67 expression and DNA synthesis.

Conclusion: By controlling p21Cip activity BRAP expression controls cell cycle activity and prevents developmental arrest in developing cardiomyocytes and apoptosis in adult cardiomyocytes.
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http://dx.doi.org/10.1093/cvr/cvz177DOI Listing
March 2020

Genetic variation in causes bradycardia by augmenting the cholinergic response via increased acetylcholine-activated potassium current ().

Dis Model Mech 2019 07 9;12(7). Epub 2019 Jul 9.

Amsterdam UMC, University of Amsterdam, Department of Experimental Cardiology, Heart Center, 1105 AZ Amsterdam, The Netherlands

Mutations in , encoding the G-protein β5 subunit (Gβ5), have recently been linked to a multisystem disorder that includes severe bradycardia. Here, we investigated the mechanism underlying bradycardia caused by the recessive p.S81L Gβ5 variant. Using CRISPR/Cas9-based targeting, we generated an isogenic series of human induced pluripotent stem cell (hiPSC) lines that were either wild type, heterozygous or homozygous for the p.S81L variant. These were differentiated into cardiomyocytes (hiPSC-CMs) that robustly expressed the acetylcholine-activated potassium channel [I(KACh); also known as I]. Baseline electrophysiological properties of the lines did not differ. Upon application of carbachol (CCh), homozygous p.S81L hiPSC-CMs displayed an increased acetylcholine-activated potassium current () density and a more pronounced decrease of spontaneous activity as compared to wild-type and heterozygous p.S81L hiPSC-CMs, explaining the bradycardia in homozygous carriers. Application of the specific I(KACh) blocker XEN-R0703 resulted in near-complete reversal of the phenotype. Our results provide mechanistic insights and proof of principle for potential therapy in patients carrying mutations.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/dmm.037994DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6679373PMC
July 2019

Pak2 as a Novel Therapeutic Target for Cardioprotective Endoplasmic Reticulum Stress Response.

Circ Res 2019 03;124(5):696-711

From the Faculty of Biology, Medicine and Health, The University of Manchester, United Kingdom (P.B., S.W., M.Z., L.C., S.K., Y.L., N.H., E.S., A.R., D.O., E.J.C., X.W., W.L.).

Rationale: Secreted and membrane-bound proteins, which account for 1/3 of all proteins, play critical roles in heart health and disease. The endoplasmic reticulum (ER) is the site for synthesis, folding, and quality control of these proteins. Loss of ER homeostasis and function underlies the pathogenesis of many forms of heart disease.

Objective: To investigate mechanisms responsible for regulating cardiac ER function, and to explore therapeutic potentials of strengthening ER function to treat heart disease.

Methods And Results: Screening a range of signaling molecules led to the discovery that Pak (p21-activated kinase)2 is a stress-responsive kinase localized in close proximity to the ER membrane in cardiomyocytes. We found that Pak2 cardiac deleted mice (Pak2-CKO) under tunicamycin stress or pressure overload manifested a defective ER response, cardiac dysfunction, and profound cell death. Small chemical chaperone tauroursodeoxycholic acid treatment of Pak2-CKO mice substantiated that Pak2 loss-induced cardiac damage is an ER-dependent pathology. Gene array analysis prompted a detailed mechanistic study, which revealed that Pak2 regulation of protective ER function was via the IRE (inositol-requiring enzyme)-1/XBP (X-box-binding protein)-1-dependent pathway. We further discovered that this regulation was conferred by Pak2 inhibition of PP2A (protein phosphatase 2A) activity. Moreover, IRE-1 activator, Quercetin, and adeno-associated virus serotype-9-delivered XBP-1s were able to relieve ER dysfunction in Pak2-CKO hearts. This provides functional evidence, which supports the mechanism underlying Pak2 regulation of IRE-1/XBP-1s signaling. Therapeutically, inducing Pak2 activation by genetic overexpression or adeno-associated virus serotype-9-based gene delivery was capable of strengthening ER function, improving cardiac performance, and diminishing apoptosis, thus protecting the heart from failure.

Conclusions: Our findings uncover a new cardioprotective mechanism, which promotes a protective ER stress response via the modulation of Pak2. This novel therapeutic strategy may present as a promising option for treating cardiac disease and heart failure.
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http://dx.doi.org/10.1161/CIRCRESAHA.118.312829DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6407830PMC
March 2019

The reduced activity of PP-1α under redox stress condition is a consequence of GSH-mediated transient disulfide formation.

Sci Rep 2018 12 7;8(1):17711. Epub 2018 Dec 7.

Functional Proteomics at the Faculty of Medicine, Goethe-Universität Frankfurt am Main, Frankfurt, Germany.

Heart failure is the most common cause of morbidity and hospitalization in the western civilization. Protein phosphatases play a key role in the basal cardiac contractility and in the responses to β-adrenergic stimulation with type-1 phosphatase (PP-1) being major contributor. We propose here that formation of transient disulfide bridges in PP-1α might play a leading role in oxidative stress response. First, we established an optimized workflow, the so-called "cross-over-read" search method, for the identification of disulfide-linked species using permutated databases. By applying this method, we demonstrate the formation of unexpected transient disulfides in PP-1α to shelter against over-oxidation. This protection mechanism strongly depends on the fast response in the presence of reduced glutathione. Our work points out that the dimerization of PP-1α involving Cys and Cys is presumably important for the protection of PP-1α active surface in the absence of a substrate. We finally give insight into the electron transport from the PP-1α catalytic core to the surface. Our data suggest that the formation of transient disulfides might be a general mechanism of proteins to escape from irreversible cysteine oxidation and to prevent their complete inactivation.
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http://dx.doi.org/10.1038/s41598-018-36267-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6286341PMC
December 2018

Defective Mitochondrial Cardiolipin Remodeling Dampens HIF-1α Expression in Hypoxia.

Cell Rep 2018 10;25(3):561-570.e6

Department of Cellular Biochemistry, University Medical Center Göttingen, GZMB, 37073 Göttingen, Germany.

Mitochondria fulfill vital metabolic functions and act as crucial cellular signaling hubs, integrating their metabolic status into the cellular context. Here, we show that defective cardiolipin remodeling, upon loss of the cardiolipin acyl transferase tafazzin, decreases HIF-1α signaling in hypoxia. Tafazzin deficiency does not affect posttranslational HIF-1α regulation but rather HIF-1α gene expression, a dysfunction recapitulated in iPSC-derived cardiomyocytes from Barth syndrome patients with tafazzin deficiency. RNA-seq analyses confirmed drastically altered signaling in tafazzin mutant cells. In hypoxia, tafazzin-deficient cells display reduced production of reactive oxygen species (ROS) perturbing NF-κB activation and concomitantly HIF-1α gene expression. Tafazzin-deficient mice hearts display reduced HIF-1α levels and undergo maladaptive hypertrophy with heart failure in response to pressure overload challenge. We conclude that defective mitochondrial cardiolipin remodeling dampens HIF-1α signaling due to a lack of NF-κB activation through reduced mitochondrial ROS production, decreasing HIF-1α transcription.
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http://dx.doi.org/10.1016/j.celrep.2018.09.057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6205837PMC
October 2018

Sarcoplasmic reticulum calcium leak contributes to arrhythmia but not to heart failure progression.

Sci Transl Med 2018 09;10(458)

Department of Cardiology and Pneumology, Georg-August-University, 37075 Göttingen, Germany.

Increased sarcoplasmic reticulum (SR) Ca leak via the cardiac ryanodine receptor (RyR2) has been suggested to play a mechanistic role in the development of heart failure (HF) and cardiac arrhythmia. Mice treated with a selective RyR2 stabilizer, rycal S36, showed normalization of SR Ca leak and improved survival in pressure overload (PO) and myocardial infarction (MI) models. The development of HF, measured by echocardiography and molecular markers, showed no difference in rycal S36- versus placebo-treated mice. Reduction of SR Ca leak in the PO model by the rycal-unrelated RyR2 stabilizer dantrolene did not mitigate HF progression. Development of HF was not aggravated by increased SR Ca leak due to RyR2 mutation (R2474S) in volume overload, an SR Ca leak-independent HF model. Arrhythmia episodes were reduced by rycal S36 treatment in PO and MI mice in vivo and ex vivo in Langendorff-perfused hearts. Isolated cardiomyocytes from murine failing hearts and human ventricular failing and atrial nonfailing myocardium showed reductions in delayed afterdepolarizations, in spontaneous and induced Ca waves, and in triggered activity in rycal S36 versus placebo cells, whereas the Ca transient, SR Ca load, SR Ca adenosine triphosphatase function, and action potential duration were not affected. Rycal S36 treatment of human induced pluripotent stem cells isolated from a patient with catecholaminergic polymorphic ventricular tachycardia could rescue the leaky RyR2 receptor. These results suggest that SR Ca leak does not primarily influence contractile HF progression, whereas rycal S36 treatment markedly reduces ventricular arrhythmias, thereby improving survival in mice.
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http://dx.doi.org/10.1126/scitranslmed.aan0724DOI Listing
September 2018

Deep phenotyping of human induced pluripotent stem cell-derived atrial and ventricular cardiomyocytes.

JCI Insight 2018 06 21;3(12). Epub 2018 Jun 21.

Clinic for Cardiology and Pneumology, University Medical Center Göttingen (UMG), Göttingen, Germany.

Generation of homogeneous populations of subtype-specific cardiomyocytes (CMs) derived from human induced pluripotent stem cells (iPSCs) and their comprehensive phenotyping is crucial for a better understanding of the subtype-related disease mechanisms and as tools for the development of chamber-specific drugs. The goals of this study were to apply a simple and efficient method for differentiation of iPSCs into defined functional CM subtypes in feeder-free conditions and to obtain a comprehensive understanding of the molecular, cell biological, and functional properties of atrial and ventricular iPSC-CMs on both the single-cell and engineered heart muscle (EHM) level. By a stage-specific activation of retinoic acid signaling in monolayer-based and well-defined culture, we showed that cardiac progenitors can be directed towards a highly homogeneous population of atrial CMs. By combining the transcriptome and proteome profiling of the iPSC-CM subtypes with functional characterizations via optical action potential and calcium imaging, and with contractile analyses in EHM, we demonstrated that atrial and ventricular iPSC-CMs and -EHM highly correspond to the atrial and ventricular heart muscle, respectively. This study provides a comprehensive understanding of the molecular and functional identities characteristic of atrial and ventricular iPSC-CMs and -EHM and supports their suitability in disease modeling and chamber-specific drug screening.
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http://dx.doi.org/10.1172/jci.insight.99941DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6124434PMC
June 2018

MicroRNAs in cardiomyocyte differentiation and maturation.

Cardiovasc Res 2018 05;114(6):779-781

Institute of Pharmacology and Toxicology, Medical Faculty Carl Gustav Carus, Technische Universität Dresden, Fetscherstr. 74, 01307 Dresden, Germany.

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http://dx.doi.org/10.1093/cvr/cvy065DOI Listing
May 2018

Severe DCM phenotype of patient harboring RBM20 mutation S635A can be modeled by patient-specific induced pluripotent stem cell-derived cardiomyocytes.

J Mol Cell Cardiol 2017 12 21;113:9-21. Epub 2017 Sep 21.

Department of Cardiology and Pneumology, Universitätsmedizin Göttingen, Germany; Institute of Pharmacology and Toxicology, Technische Universität Dresden, Germany. Electronic address:

The ability to generate patient-specific induced pluripotent stem cells (iPSCs) provides a unique opportunity for modeling heart disease in vitro. In this study, we generated iPSCs from a patient with dilated cardiomyopathy (DCM) caused by a missense mutation S635A in RNA-binding motif protein 20 (RBM20) and investigated the functionality and cell biology of cardiomyocytes (CMs) derived from patient-specific iPSCs (RBM20-iPSCs). The RBM20-iPSC-CMs showed abnormal distribution of sarcomeric α-actinin and defective calcium handling compared to control-iPSC-CMs, suggesting disorganized myofilament structure and altered calcium machinery in CMs of the RBM20 patient. Engineered heart muscles (EHMs) from RBM20-iPSC-CMs showed that not only active force generation was impaired in RBM20-EHMs but also passive stress of the tissue was decreased, suggesting a higher visco-elasticity of RBM20-EHMs. Furthermore, we observed a reduced titin (TTN) N2B-isoform expression in RBM20-iPSC-CMs by demonstrating a reduction of exon skipping in the PEVK region of TTN and an inhibition of TTN isoform switch. In contrast, in control-iPSC-CMs both TTN isoforms N2B and N2BA were expressed, indicating that the TTN isoform switch occurs already during early cardiogenesis. Using next generation RNA sequencing, we mapped transcriptome and splicing target profiles of RBM20-iPSC-CMs and identified different cardiac gene networks in response to the analyzed RBM20 mutation in cardiac-specific processes. These findings shed the first light on molecular mechanisms of RBM20-dependent pathological cardiac remodeling leading to DCM. Our data demonstrate that iPSC-CMs coupled with EHMs provide a powerful tool for evaluating disease-relevant functional defects and for a deeper mechanistic understanding of alternative splicing-related cardiac diseases.
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http://dx.doi.org/10.1016/j.yjmcc.2017.09.008DOI Listing
December 2017

CRISPR correction of the PRKAG2 gene mutation in the patient's induced pluripotent stem cell-derived cardiomyocytes eliminates electrophysiological and structural abnormalities.

Heart Rhythm 2018 02 14;15(2):267-276. Epub 2017 Sep 14.

Department of Physiology, Biophysics and Systems Biology, Technion, Haifa, Israel; The Rappaport Institute, Technion, Haifa, Israel; Rappaport Faculty of Medicine, Technion, Haifa, Israel. Electronic address:

Background: Mutations in the PRKAG2 gene encoding the γ-subunit of adenosine monophosphate kinase (AMPK) cause hypertrophic cardiomyopathy (HCM) and familial Wolff-Parkinson-White (WPW) syndrome. Patients carrying the R302Q mutation in PRKAG2 present with sinus bradycardia, escape rhythms, ventricular preexcitation, supraventricular tachycardia, and atrioventricular block. This mutation affects AMPK activity and increases glycogen storage in cardiomyocytes. The link between glycogen storage, WPW syndrome, HCM, and arrhythmias remains unknown.

Objective: The purpose of this study was to investigate the pathological changes caused by the PRKAG2 mutation. We tested the hypothesis that patient's induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) display clinical aspects of the disease.

Methods: Using clustered regularly interspaced short palindromic repeats (CRISPR) technology, we corrected the mutation and then generated isogenic iPSC-CMs. Action potentials were recorded from spontaneously firing and paced cardiomyocytes using the patch clamp technique. Using a microelectrode array setup, we recorded electrograms from iPSC-CMs clusters. Transmission electron microscopy was used to detect ultrastructural abnormalities in the mutated iPSC-CMs.

Results: PRKAG2-mutated iPSC-CMs exhibited abnormal firing patterns, delayed afterdepolarizations, triggered arrhythmias, and augmented beat rate variability. Importantly, CRISPR correction eliminated the electrophysiological abnormalities, the augmented glycogen, storage, and cardiomyocyte hypertrophy.

Conclusion: PRKAG2-mutated iPSC-CMs displayed functional and structural abnormalities, which were abolished by correcting the mutation in the patient's iPSCs using CRISPR technology.
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http://dx.doi.org/10.1016/j.hrthm.2017.09.024DOI Listing
February 2018

Efficient Killing of Murine Pluripotent Stem Cells by Natural Killer (NK) Cells Requires Activation by Cytokines and Partly Depends on the Activating NK Receptor NKG2D.

Front Immunol 2017 26;8:870. Epub 2017 Jul 26.

Institute of Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany.

Natural killer (NK) cells play an important role as cytotoxic effector cells, which scan the organism for infected or tumorigenic cells. Conflicting data have been published whether NK cells can also kill allogeneic or even autologous pluripotent stem cells (PSCs) and which receptors are involved. A clarification of this question is relevant since an activity of NK cells against PSCs could reduce the risk of teratoma growth after transplantation of PSC-derived grafts. Therefore, the hypothesis has been tested that the activity of NK cells against PSCs depends on cytokine activation and specifically on the activating NK receptor NKG2D. It is shown that a subcutaneous injection of autologous iPSCs failed to activate NK cells against these iPSCs and can give rise to teratomas. In agreement with this result, several PSC lines, including two iPSC, two embryonic stem cell (ESC), and two so-called multipotent adult germline stem cell (maGSC) lines, were largely resistant against resting NK cells although differences in killing were found at low level. All PSC lines were killed by interleukin (IL)-2-activated NK cells, and maGSCs were better killed than the other PSC types. The PSCs expressed ligands of the activating NK receptor NKG2D and NKG2D-deficient NK cells from mice were impaired in their cytotoxic activity against PSCs. The low-cytotoxic activity of resting NK cells was almost completely dependent on NKG2D. The cytotoxic activity of IL-2-activated NKG2D-deficient NK cells against PSCs was reduced, indicating that also other activating receptors on cytokine-activated NK cells must be engaged by ligands on PSCs. Thus, NKG2D is an important activating receptor involved in killing of murine PSCs. However, NK cells need to be activated by cytokines before they efficiently target PSCs and then also other NK receptors become relevant. These features of NK cells might be relevant for transplantation of PSC-derived grafts since NK cells have the capability to kill undifferentiated cells, which might be present in grafts in trace amounts.
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http://dx.doi.org/10.3389/fimmu.2017.00870DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5582315PMC
July 2017

Catecholamine-Dependent β-Adrenergic Signaling in a Pluripotent Stem Cell Model of Takotsubo Cardiomyopathy.

J Am Coll Cardiol 2017 Aug;70(8):975-991

Clinic for Cardiology and Pneumology, University Medical Center Göttingen, Göttingen, Germany. Electronic address:

Background: Takotsubo syndrome (TTS) is characterized by an acute left ventricular dysfunction and is associated with life-threating complications in the acute phase. The underlying disease mechanism in TTS is still unknown. A genetic basis has been suggested to be involved in the pathogenesis.

Objectives: The aims of the study were to establish an in vitro induced pluripotent stem cell (iPSC) model of TTS, to test the hypothesis of altered β-adrenergic signaling in TTS iPSC-cardiomyocytes (CMs), and to explore whether genetic susceptibility underlies the pathophysiology of TTS.

Methods: Somatic cells of patients with TTS and control subjects were reprogrammed to iPSCs and differentiated into CMs. Three-month-old CMs were subjected to catecholamine stimulation to simulate neurohumoral overstimulation. We investigated β-adrenergic signaling and TTS cardiomyocyte function.

Results: Enhanced β-adrenergic signaling in TTS-iPSC-CMs under catecholamine-induced stress increased expression of the cardiac stress marker NR4A1; cyclic adenosine monophosphate levels; and cyclic adenosine monophosphate-dependent protein kinase A-mediated hyperphosphorylation of RYR2-S2808, PLN-S16, TNI-S23/24, and Cav1.2-S1928, and leads to a reduced calcium time to transient 50% decay. These cellular catecholamine-dependent responses were mainly mediated by β-adrenoceptor signaling in TTS. Engineered heart muscles from TTS-iPSC-CMs showed an impaired force of contraction and a higher sensitivity to isoprenaline-stimulated inotropy compared with control subjects. In addition, altered electrical activity and increased lipid accumulation were detected in catecholamine-treated TTS-iPSC-CMs, and were confirmed by differentially expressed lipid transporters CD36 and CPT1C. Furthermore, we uncovered genetic variants in different key regulators of cardiac function.

Conclusions: Enhanced β-adrenergic signaling and higher sensitivity to catecholamine-induced toxicity were identified as mechanisms associated with the TTS phenotype. (International Takotsubo Registry [InterTAK Registry] [InterTAK]; NCT01947621).
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http://dx.doi.org/10.1016/j.jacc.2017.06.061DOI Listing
August 2017