Publications by authors named "Malte Tiburcy"

42 Publications

Generation of Engineered Human Myocardium in a Multi-well Format.

STAR Protoc 2020 Jun 3;1(1):100032. Epub 2020 Jun 3.

Institute of Pharmacology and Toxicology, University Medical Center Göttingen, Göttingen, Germany.

This protocol describes a robust method for the generation of engineered human myocardium (EHM) from pluripotent stem cells (PSCs) in a multi-well plate under defined, serum-free conditions. By parallel culture of up to 48 EHM in one plate, contractile heart muscle can be obtained to serve numerous applications, including drug screening and disease modelling. This protocol has been successfully applied to human embryonic stem (HES) cell- and induced PSC-derived cardiomyocytes, subtype-specific, i.e., atrial and ventricular, and commercially available cardiomyocyte preparations. For complete details on the use and execution of this protocol, please refer to Tiburcy et al. (2017).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.xpro.2020.100032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7580201PMC
June 2020

Inhibition of Prolyl-Hydroxylase Domain Enzymes Protects From Reoxygenation Injury in Engineered Human Myocardium.

Circulation 2020 Oct 26;142(17):1694-1696. Epub 2020 Oct 26.

Institute of Pharmacology and Toxicology, University Medical Center Göttingen, Germany (E.L., C.N., L.C.Z., W-H.Z., M.T.).

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCULATIONAHA.119.044471DOI Listing
October 2020

Inhibition of aquaporin-1 prevents myocardial remodeling by blocking the transmembrane transport of hydrogen peroxide.

Sci Transl Med 2020 10;12(564)

Institute of Experimental and Clinical Research (IREC), Pharmacology and Therapeutics (FATH), Cliniques Universitaires St Luc and Université Catholique de Louvain (UCLouvain), 1200 Brussels, Belgium.

Pathological remodeling of the myocardium has long been known to involve oxidant signaling, but strategies using systemic antioxidants have generally failed to prevent it. We sought to identify key regulators of oxidant-mediated cardiac hypertrophy amenable to targeted pharmacological therapy. Specific isoforms of the aquaporin water channels have been implicated in oxidant sensing, but their role in heart muscle is unknown. RNA sequencing from human cardiac myocytes revealed that the archetypal is a major isoform. expression correlates with the severity of hypertrophic remodeling in patients with aortic stenosis. The AQP1 channel was detected at the plasma membrane of human and mouse cardiac myocytes from hypertrophic hearts, where it colocalized with NADPH oxidase-2 and caveolin-3. We show that hydrogen peroxide (HO), produced extracellularly, is necessary for the hypertrophic response of isolated cardiac myocytes and that AQP1 facilitates the transmembrane transport of HO through its water pore, resulting in activation of oxidant-sensitive kinases in cardiac myocytes. Structural analysis of the amino acid residues lining the water pore of AQP1 supports its permeation by HO Deletion of or selective blockade of the AQP1 intrasubunit pore inhibited HO transport in mouse and human cells and rescued the myocyte hypertrophy in human induced pluripotent stem cell-derived engineered heart muscle. Treatment of mice with a clinically approved AQP1 inhibitor, Bacopaside, attenuated cardiac hypertrophy. We conclude that cardiac hypertrophy is mediated by the transmembrane transport of HO by the water channel AQP1 and that inhibitors of AQP1 represent new possibilities for treating hypertrophic cardiomyopathies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/scitranslmed.aay2176DOI Listing
October 2020

Intronic CRISPR Repair in a Preclinical Model of Noonan Syndrome-Associated Cardiomyopathy.

Circulation 2020 Sep 6;142(11):1059-1076. Epub 2020 Jul 6.

Clinic for Cardiology and Pneumology (U.H., M.K., L.R., R.H., G.H., L.C.).

Background: Noonan syndrome (NS) is a multisystemic developmental disorder characterized by common, clinically variable symptoms, such as typical facial dysmorphisms, short stature, developmental delay, intellectual disability as well as cardiac hypertrophy. The underlying mechanism is a gain-of-function of the RAS-mitogen-activated protein kinase signaling pathway. However, our understanding of the pathophysiological alterations and mechanisms, especially of the associated cardiomyopathy, remains limited and effective therapeutic options are lacking.

Methods: Here, we present a family with two siblings displaying an autosomal recessive form of NS with massive hypertrophic cardiomyopathy as clinically the most prevalent symptom caused by biallelic mutations within the leucine zipper-like transcription regulator 1 (). We generated induced pluripotent stem cell-derived cardiomyocytes of the affected siblings and investigated the patient-specific cardiomyocytes on the molecular and functional level.

Results: Patients' induced pluripotent stem cell-derived cardiomyocytes recapitulated the hypertrophic phenotype and uncovered a so-far-not-described causal link between LZTR1 dysfunction, RAS-mitogen-activated protein kinase signaling hyperactivity, hypertrophic gene response and cellular hypertrophy. Calcium channel blockade and MEK inhibition could prevent some of the disease characteristics, providing a molecular underpinning for the clinical use of these drugs in patients with NS, but might not be a sustainable therapeutic option. In a proof-of-concept approach, we explored a clinically translatable intronic CRISPR (clustered regularly interspaced short palindromic repeats) repair and demonstrated a rescue of the hypertrophic phenotype.

Conclusions: Our study revealed the human cardiac pathogenesis in patient-specific induced pluripotent stem cell-derived cardiomyocytes from NS patients carrying biallelic variants in and identified a unique disease-specific proteome signature. In addition, we identified the intronic CRISPR repair as a personalized and in our view clinically translatable therapeutic strategy to treat NS-associated hypertrophic cardiomyopathy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCULATIONAHA.119.044794DOI Listing
September 2020

Non-Human Primate iPSC Generation, Cultivation, and Cardiac Differentiation under Chemically Defined Conditions.

Cells 2020 05 29;9(6). Epub 2020 May 29.

Research Platform Degenerative Diseases, German Primate Center-Leibniz Institute for Primate Research, Kellnerweg 4, 37077 Göttingen, Germany.

Non-human primates (NHP) are important surrogate models for late preclinical development of advanced therapy medicinal products (ATMPs), including induced pluripotent stem cell (iPSC)-based therapies, which are also under development for heart failure repair. For effective heart repair by remuscularization, large numbers of cardiomyocytes are required, which can be obtained by efficient differentiation of iPSCs. However, NHP-iPSC generation and long-term culture in an undifferentiated state under feeder cell-free conditions turned out to be problematic. Here we describe the reproducible development of rhesus macaque () iPSC lines. Postnatal rhesus skin fibroblasts were reprogrammed under chemically defined conditions using non-integrating vectors. The robustness of the protocol was confirmed using another NHP species, the olive baboon (). Feeder-free maintenance of NHP-iPSCs was essentially dependent on concurrent Wnt-activation by GSK-inhibition (Gi) and Wnt-inhibition (Wi). Generated NHP-iPSCs were successfully differentiated into cardiomyocytes using a combined growth factor/GiWi protocol. The capacity of the iPSC-derived cardiomyocytes to self-organize into contractile engineered heart muscle (EHM) was demonstrated. Collectively, this study establishes a reproducible protocol for the robust generation and culture of NHP-iPSCs, which are useful for preclinical testing of strategies for cell replacement therapies in NHP.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cells9061349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7349583PMC
May 2020

Long-term human IgG treatment improves heart and muscle function in a mouse model of Duchenne muscular dystrophy.

J Cachexia Sarcopenia Muscle 2020 08 20;11(4):1018-1031. Epub 2020 May 20.

Department of Neurology, University Medical Center Göttingen, Göttingen, Germany.

Background: Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease caused by mutations in the dystrophin gene, which leads to structural instability of the dystrophin-glycoprotein-complex with subsequent muscle degeneration. In addition, muscle inflammation has been implicated in disease progression and therapeutically addressed with glucocorticosteroids. These have numerous adverse effects. Treatment with human immunoglobulin G (IgG) improved clinical and para-clinical parameters in the early disease phase in the well-established mdx mouse model. The aim of the present study was to confirm the efficacy of IgG in a long-term pre-clinical study in mdx mice.

Methods: IgG (2 g/kg body weight) or NaCl solution as control was administered monthly over 18 months by intraperitoneal injection in mdx mice beginning at 3 weeks of age. Several clinical outcome measures including endurance, muscle strength, and echocardiography were assessed. After 18 months, the animals were sacrificed, blood was collected for analysis, and muscle samples were obtained for ex vivo muscle contraction tests, quantitative PCR, and histology.

Results: IgG significantly improved the daily voluntary running performance (1.9 m more total daily running distance, P < 0.0001) and slowed the decrease in grip strength by 0.1 mN, (P = 0.018). IgG reduced fatigability of the diaphragm (improved ratio to maximum force by 0.09 ± 0.04, P = 0.044), but specific tetanic force remained unchanged in the ex vivo muscle contraction test. Cardiac function was significantly better after IgG, especially fractional area shortening (P = 0.012). These results were accompanied by a reduction in cardiac fibrosis and the infiltration of T cells (P = 0.0002) and macrophages (P = 0.0027). In addition, treatment with IgG resulted in a significant reduction of the infiltration of T cells (P ≤ 0.036) in the diaphragm, gastrocnemius, quadriceps, and a similar trend in tibialis anterior and macrophages (P ≤ 0.045) in gastrocnemius, quadriceps, tibialis anterior, and a similar trend in the diaphragm, as well as a decrease in myopathic changes as reflected by a reduced central nuclear index in the diaphragm, tibialis anterior, and quadriceps (P ≤ 0.002 in all).

Conclusions: The present study underscores the importance of an inflammatory contribution to the disease progression of DMD. The data demonstrate the long-term efficacy of IgG in the mdx mouse. IgG is well tolerated by humans and could preferentially complement gene therapy in DMD. The data call for a clinical trial with IgG in DMD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jcsm.12569DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7432639PMC
August 2020

CRISPLD1: a novel conserved target in the transition to human heart failure.

Basic Res Cardiol 2020 03 7;115(3):27. Epub 2020 Mar 7.

Laboratory of Experimental Cardiology, Clinic for Cardiology and Pneumology, Heart Research Center, University Medical Center Goettingen, Robert-Koch-Str. 40, 37075, Göttingen, Germany.

Heart failure is a major health problem worldwide with a significant morbidity and mortality rate. Although studied extensively in animal models, data from patients at the compensated disease stage are lacking. We sampled myocardium biopsies from aortic stenosis patients with compensated hypertrophy and moderate heart failure and used transcriptomics to study the transition to failure. Sequencing and comparative analysis of analogous samples of mice with transverse aortic constriction identified 25 candidate genes with similar regulation in response to pressure overload, reflecting highly conserved molecular processes. The gene cysteine-rich secretory protein LCCL domain containing 1 (CRISPLD1) is upregulated in the transition to failure in human and mouse and its function is unknown. Homology to ion channel regulatory toxins suggests a role in Ca cycling. CRISPR/Cas9-mediated loss-of-function leads to dysregulated Ca handling in human-induced pluripotent stem cell-derived cardiomyocytes. The downregulation of prohypertrophic, proapoptotic and Ca-signaling pathways upon CRISPLD1-KO and its upregulation in the transition to failure implicates a contribution to adverse remodeling. These findings provide new pathophysiological data on Ca regulation in the transition to failure and novel candidate genes with promising potential for therapeutic interventions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00395-020-0784-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060963PMC
March 2020

Regeneration competent satellite cell niches in rat engineered skeletal muscle.

FASEB Bioadv 2019 Dec 13;1(12):731-746. Epub 2019 Nov 13.

Institute of Pharmacology and Toxicology Georg-August University Göttingen Göttingen Germany.

Satellite cells reside in defined niches and are activated upon skeletal muscle injury to facilitate regeneration. Mechanistic studies of skeletal muscle regeneration are hampered by the inability to faithfully simulate satellite cell biology in vitro. We sought to overcome this limitation by developing tissue engineered skeletal muscle (ESM) with (1) satellite cell niches and (2) the capacity to regenerate after injury. ESMs contained quiescent Pax7-positive satellite cells in morphologically defined niches. Satellite cells could be activated to repair (i) cardiotoxin and (ii) mechanical crush injuries. Activation of the Wnt-pathway was essential for muscle regeneration. Finally, muscle progenitors from the engineered niche developed de novo ESM in vitro and regenerated skeletal muscle after cardiotoxin-induced injury in vivo. We conclude that ESM with functional progenitor niches reminiscent of the in vivo satellite cell niches can be engineered in vitro. ESM may ultimately be exploited in disease modeling, drug screening, or muscle regeneration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fba.2019-00013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6996344PMC
December 2019

Troponin destabilization impairs sarcomere-cytoskeleton interactions in iPSC-derived cardiomyocytes from dilated cardiomyopathy patients.

Sci Rep 2020 01 14;10(1):209. Epub 2020 Jan 14.

Heart Center, Department of Cardiology and Pneumology, Goettingen, Germany.

The sarcomeric troponin-tropomyosin complex is a critical mediator of excitation-contraction coupling, sarcomeric stability and force generation. We previously reported that induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from patients with a dilated cardiomyopathy (DCM) mutation, troponin T (TnT)-R173W, display sarcomere protein misalignment and impaired contractility. Yet it is not known how TnT mutation causes dysfunction of sarcomere microdomains and how these events contribute to misalignment of sarcomeric proteins in presence of DCM TnT-R173W. Using a human iPSC-CM model combined with CRISPR/Cas9-engineered isogenic controls, we uncovered that TnT-R173W destabilizes molecular interactions of troponin with tropomyosin, and limits binding of PKA to local sarcomere microdomains. This attenuates troponin phosphorylation and dysregulates local sarcomeric microdomains in DCM iPSC-CMs. Disrupted microdomain signaling impairs MYH7-mediated, AMPK-dependent sarcomere-cytoskeleton filament interactions and plasma membrane attachment. Small molecule-based activation of AMPK can restore TnT microdomain interactions, and partially recovers sarcomere protein misalignment as well as impaired contractility in DCM TnT-R173W iPSC-CMs. Our findings suggest a novel therapeutic direction targeting sarcomere- cytoskeleton interactions to induce sarcomere re-organization and contractile recovery in DCM.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-56597-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6959358PMC
January 2020

Engineered heart tissue models from hiPSC-derived cardiomyocytes and cardiac ECM for disease modeling and drug testing applications.

Acta Biomater 2019 07 7;92:145-159. Epub 2019 May 7.

Sohnis Research Laboratory for Cardiac Electrophysiology and Regenerative Medicine, The Rappaport Faculty of Medicine and Research Institute, Technion - Israel Institute of Technology, Israel; Cardiology Department, Rambam Health Care Campus, Israel. Electronic address:

Cardiac tissue engineering provides unique opportunities for cardiovascular disease modeling, drug testing, and regenerative medicine applications. To recapitulate human heart tissue, we combined human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with a chitosan-enhanced extracellular-matrix (ECM) hydrogel, derived from decellularized pig hearts. Ultrastructural characterization of the ECM-derived engineered heart tissues (ECM-EHTs) revealed an anisotropic muscle structure, with embedded cardiomyocytes showing more mature properties than 2D-cultured hiPSC-CMs. Force measurements confirmed typical force-length relationships, sensitivity to extracellular calcium, and adequate ionotropic responses to contractility modulators. By combining genetically-encoded calcium and voltage indicators with laser-confocal microscopy and optical mapping, the electrophysiological and calcium-handling properties of the ECM-EHTs could be studied at the cellular and tissue resolutions. This allowed to detect drug-induced changes in contraction rate (isoproterenol, carbamylcholine), optical signal morphology (E-4031, ATX2, isoproterenol, ouabin and quinidine), cellular arrhythmogenicity (E-4031 and ouabin) and alterations in tissue conduction properties (lidocaine, carbenoxolone and quinidine). Similar assays in ECM-EHTs derived from patient-specific hiPSC-CMs recapitulated the abnormal phenotype of the long QT syndrome and catecholaminergic polymorphic ventricular tachycardia. Finally, programmed electrical stimulation and drug-induced pro-arrhythmia led to the development of reentrant arrhythmias in the ECM-EHTs. In conclusion, a novel ECM-EHT model was established, which can be subjected to high-resolution long-term serial functional phenotyping, with important implications for cardiac disease modeling, drug testing and precision medicine. STATEMENT OF SIGNIFICANCE: One of the main objectives of cardiac tissue engineering is to create an in-vitro muscle tissue surrogate of human heart tissue. To this end, we combined a chitosan-enforced cardiac-specific ECM hydrogel derived from decellularized pig hearts with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from healthy-controls and patients with inherited cardiac disorders. We then utilized genetically-encoded calcium and voltage fluorescent indicators coupled with unique optical imaging techniques and force-measurements to study the functional properties of the generated engineered heart tissues (EHTs). These studies demonstrate the unique potential of the new model for physiological and pathophysiological studies (assessing contractility, conduction and reentrant arrhythmias), novel disease modeling strategies ("disease-in-a-dish" approach) for studying inherited arrhythmogenic disorders, and for drug testing applications (safety pharmacology).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.actbio.2019.05.016DOI Listing
July 2019

Cardiac macrotissues-on-a-plate models for phenotypic drug screens.

Adv Drug Deliv Rev 2019 02 19;140:93-100. Epub 2019 Mar 19.

Institute of Pharmacology and Toxicology, University Medical Center, Georg-August-University, Göttingen, Germany; DZHK (German Center for Cardiovascular Research), Göttingen, Germany. Electronic address:

Facilitated by the introduction of human induced pluripotent stem cells and protocols for their efficient directed differentiation at high quantity and quality, innovative human heart muscle models are being developed for applications in drug screens. Employed models range from the microscopic cardiomyocytes-on-a-chip scale to the cardiac macrotissues-on-a-plate scale. Whilst cardiomyocyte-on-a-chip models can be readily adapted to high-throughput primary screening, they are limited as to the deep phenotyping of contractility, and here in particular contractile force development. In lower throughput cardiac macrotissue-on-a-plate platforms, organotypic function, including anisotropic electrical spread of excitation and contractility, can be recapitulated at the macroscopic scale. This review serves as an overview of cardiac macrotissue-on-a-plate technologies with a focus on their application in the investigation of drug effects on heart muscle contractility and disease modeling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.addr.2019.03.002DOI Listing
February 2019

Agonistic and antagonistic roles of fibroblasts and cardiomyocytes on viscoelastic stiffening of engineered human myocardium.

Prog Biophys Mol Biol 2019 07 12;144:51-60. Epub 2018 Dec 12.

University of Göttingen, 3rd Institute of Physics - Biophysics, 37077, Göttingen, Germany. Electronic address:

Cardiomyocyte and stroma cell cross-talk is essential for the formation of collagen-based engineered heart muscle, including engineered human myocardium (EHM). Fibroblasts are a main component of the myocardial stroma. We hypothesize that fibroblasts, by compacting the surrounding collagen network, support the self-organization of cardiomyocytes into a functional syncytium. With a focus on early self-organization processes in EHM, we studied the molecular and biophysical adaptations mediated by defined populations of fibroblasts and embryonic stem cell-derived cardiomyocytes in a collagen type I hydrogel. After a short phase of cell-independent collagen gelation (30 min), tissue compaction was progressively mediated by fibroblasts. Fibroblast-mediated tissue stiffening was attenuated in the presence of cardiomyocytes allowing for the assembly of stably contracting, force-generating EHM within 4 weeks. Comparative RNA-sequencing data corroborated that fibroblasts are particularly sensitive to the tissue compaction process, resulting in the fast activation of transcription profiles, supporting heart muscle development and extracellular matrix synthesis. Large amplitude oscillatory shear (LAOS) measurements revealed nonlinear strain stiffening at physiological strain amplitudes (>2%), which was reduced in the presence of cells. The nonlinear stress-strain response could be characterized by a mathematical model. Collectively, our study defines the interplay between fibroblasts and cardiomyocytes during human heart muscle self-organization in vitro and underscores the relevance of fibroblasts in the biological engineering of a cardiomyogenesis-supporting viscoelastic stroma. We anticipate that the established mathematical model will facilitate future attempts to optimize EHM for in vitro (disease modelling) and in vivo applications (heart repair).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.pbiomolbio.2018.11.011DOI Listing
July 2019

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/jci.insight.99941DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6124434PMC
June 2018

X-ray diffraction imaging of cardiac cells and tissue.

Prog Biophys Mol Biol 2019 07 18;144:151-165. Epub 2018 Jun 18.

Universität Göttingen, Institut für Röntgenphysik, Friedrich-Hund-Platz 1, 37077 Göttingen, Germany. Electronic address:

With the development of advanced focusing optics for x-rays, we can now use x-ray beams with spot sizes in the micro- or nanometer range to scan cells and large areas of tissues and continuously record the diffraction signals. From this data, x-ray scattering maps or so-called x-ray darkfield images are computed showing how different types of cells or regions of tissues differ in their diffraction intensity. At the same time a diffraction pattern is available for each scan point which encodes the local nanostructure, averaged over many contributing constituents illuminated by the beam. In this work we have exploited these new capabilities of scanning x-ray diffraction to investigate cardiac muscle cells as well as cardiac tissue. We give examples of how cardiac cells, especially living, cultured cells, can be prepared to be compatible with the instrumentation constraints of nano- or micro-diffraction instruments. Furthermore, we show how the developmental stage, ranging from neonatal to adult cells, as well as the final preparation state of the cardiomyocytes influences the recorded scattering signal and how these diffraction signals compare to the structure of a fully developed cardiac muscle.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.pbiomolbio.2018.05.012DOI Listing
July 2019

Electrical dysfunctions in human-induced pluripotent stem cell-derived cardiomyocytes from a patient with an arrhythmogenic right ventricular cardiomyopathy.

Europace 2018 06;20(FI1):f46-f56

First Department of Medicine, Faculty of Medicine, University Medical Centre Mannheim (UMM), University of Heidelberg, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany.

Aims: Our aim is to investigate the arrhythmogenic mechanism in arrhythmogenic right ventricular cardiomyopathy (ARVC)-patients by using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs).

Methods And Results: Human-induced pluripotent stem cell-derived cardiomyocytes were generated from human skin fibroblasts of two healthy donors and an ARVC-patient with a desmoglein-2 (DSG2) mutation. Patch clamp, quantitative polymerase chain reaction, and calcium imaging techniques were employed for the study. The amplitude and maximal upstroke velocity (Vmax) of action potential (AP) in ARVC-cells were smaller than that in healthy donor cells, whereas the resting potential and AP duration (APD) was not changed. The reduced Vmax resulted from decreased peak sodium current. The reason for undetected changes in APD may be the counter-action of reduced transient outward, small conductance Ca2+-activated, adenosine triphosphate-sensitive, Na/Ca exchanger (INCX) currents, and enhanced rapidly delayed rectifier currents. Isoprenaline (Iso) reduced INCX and shortened APD in both donor and ARVC-hiPSC-CMs. However, the effects of Iso in ARVC-cells are significantly larger than that in donor cells. In addition, ARVC-hiPSC-CMs showed more frequently than donor cells arrhythmogenic events induced by adrenergic stimulation.

Conclusion: Cardiomyocytes derived from the ARVC patient with a DSG2 mutation displayed multiple ion channel dysfunctions and abnormal cellular electrophysiology as well as enhanced sensitivity to adrenergic stimulation. These may underlie the arrhythmogenesis in ARVC patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/europace/euy042DOI Listing
June 2018

Ion Channel Expression and Characterization in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Stem Cells Int 2018 8;2018:6067096. Epub 2018 Jan 8.

First Department of Medicine, Faculty of Medicine, University Medical Centre Mannheim (UMM), University of Heidelberg, Mannheim, Germany.

Background: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are providing new possibilities for the biological study, cell therapies, and drug discovery. However, the ion channel expression and functions as well as regulations in hiPSC-CMs still need to be fully characterized.

Methods: Cardiomyocytes were derived from hiPS cells that were generated from two healthy donors. qPCR and patch clamp techniques were used for the study.

Results: In addition to the reported ion channels, I, I, I, I, I, I, I, I, I I, I, I, and I, we detected both the expression and currents of ACh-activated (KACh) and Na-activated (KNa) K, volume-regulated and calcium-activated (Cl-Ca) Cl, and TRPV channels. All the detected ion currents except I, I, I, I, and TRPV1 currents contribute to AP duration. Isoprenaline increased I, I, and I but reduced I and I, without an effect on I, I, I, I, I, I, I, I, and I. Carbachol alone showed no effect on the tested ion channel currents.

Conclusion: Our data demonstrate that most ion channels, which are present in healthy or diseased cardiomyocytes, exist in hiPSC-CMs. Some of them contribute to action potential performance and are regulated by adrenergic stimulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1155/2018/6067096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5835237PMC
January 2018

Correction of diverse muscular dystrophy mutations in human engineered heart muscle by single-site genome editing.

Sci Adv 2018 01 31;4(1):eaap9004. Epub 2018 Jan 31.

Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

Genome editing with CRISPR/Cas9 is a promising new approach for correcting or mitigating disease-causing mutations. Duchenne muscular dystrophy (DMD) is associated with lethal degeneration of cardiac and skeletal muscle caused by more than 3000 different mutations in the X-linked dystrophin gene (). Most of these mutations are clustered in "hotspots." There is a fortuitous correspondence between the eukaryotic splice acceptor and splice donor sequences and the protospacer adjacent motif sequences that govern prokaryotic CRISPR/Cas9 target gene recognition and cleavage. Taking advantage of this correspondence, we screened for optimal guide RNAs capable of introducing insertion/deletion (indel) mutations by nonhomologous end joining that abolish conserved RNA splice sites in 12 exons that potentially allow skipping of the most common mutant or out-of-frame exons within or nearby mutational hotspots. We refer to the correction of DMD mutations by exon skipping as myoediting. In proof-of-concept studies, we performed myoediting in representative induced pluripotent stem cells from multiple patients with large deletions, point mutations, or duplications within the gene and efficiently restored dystrophin protein expression in derivative cardiomyocytes. In three-dimensional engineered heart muscle (EHM), myoediting of DMD mutations restored dystrophin expression and the corresponding mechanical force of contraction. Correcting only a subset of cardiomyocytes (30 to 50%) was sufficient to rescue the mutant EHM phenotype to near-normal control levels. We conclude that abolishing conserved RNA splicing acceptor/donor sites and directing the splicing machinery to skip mutant or out-of-frame exons through myoediting allow correction of the cardiac abnormalities associated with DMD by eliminating the underlying genetic basis of the disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/sciadv.aap9004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5796795PMC
January 2018

Passive Stretch Induces Structural and Functional Maturation of Engineered Heart Muscle as Predicted by Computational Modeling.

Stem Cells 2018 02 13;36(2):265-277. Epub 2017 Nov 13.

Stanford Cardiovascular Institute, Stanford University, Stanford, California, USA.

The ability to differentiate human pluripotent stem cells (hPSCs) into cardiomyocytes (CMs) makes them an attractive source for repairing injured myocardium, disease modeling, and drug testing. Although current differentiation protocols yield hPSC-CMs to >90% efficiency, hPSC-CMs exhibit immature characteristics. With the goal of overcoming this limitation, we tested the effects of varying passive stretch on engineered heart muscle (EHM) structural and functional maturation, guided by computational modeling. Human embryonic stem cells (hESCs, H7 line) or human induced pluripotent stem cells (IMR-90 line) were differentiated to hPSC-derived cardiomyocytes (hPSC-CMs) in vitro using a small molecule based protocol. hPSC-CMs were characterized by troponin flow cytometry as well as electrophysiological measurements. Afterwards, 1.2 × 10 hPSC-CMs were mixed with 0.4 × 10 human fibroblasts (IMR-90 line) (3:1 ratio) and type-I collagen. The blend was cast into custom-made 12-mm long polydimethylsiloxane reservoirs to vary nominal passive stretch of EHMs to 5, 7, or 9 mm. EHM characteristics were monitored for up to 50 days, with EHMs having a passive stretch of 7 mm giving the most consistent formation. Based on our initial macroscopic observations of EHM formation, we created a computational model that predicts the stress distribution throughout EHMs, which is a function of cellular composition, cellular ratio, and geometry. Based on this predictive modeling, we show cell alignment by immunohistochemistry and coordinated calcium waves by calcium imaging. Furthermore, coordinated calcium waves and mechanical contractions were apparent throughout entire EHMs. The stiffness and active forces of hPSC-derived EHMs are comparable with rat neonatal cardiomyocyte-derived EHMs. Three-dimensional EHMs display increased expression of mature cardiomyocyte genes including sarcomeric protein troponin-T, calcium and potassium ion channels, β-adrenergic receptors, and t-tubule protein caveolin-3. Passive stretch affects the structural and functional maturation of EHMs. Based on our predictive computational modeling, we show how to optimize cell alignment and calcium dynamics within EHMs. These findings provide a basis for the rational design of EHMs, which enables future scale-up productions for clinical use in cardiovascular tissue engineering. Stem Cells 2018;36:265-277.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/stem.2732DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5785460PMC
February 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.yjmcc.2017.09.008DOI Listing
December 2017

Functional correction of dystrophin actin binding domain mutations by genome editing.

JCI Insight 2017 09 21;2(18). Epub 2017 Sep 21.

Department of Molecular Biology.

Dystrophin maintains the integrity of striated muscles by linking the actin cytoskeleton with the cell membrane. Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene (DMD) that result in progressive, debilitating muscle weakness, cardiomyopathy, and a shortened lifespan. Mutations of dystrophin that disrupt the amino-terminal actin-binding domain 1 (ABD-1), encoded by exons 2-8, represent the second-most common cause of DMD. In the present study, we compared three different strategies for CRISPR/Cas9 genome editing to correct mutations in the ABD-1 region of the DMD gene by deleting exons 3-9, 6-9, or 7-11 in human induced pluripotent stem cells (iPSCs) and by assessing the function of iPSC-derived cardiomyocytes. All three exon deletion strategies enabled the expression of truncated dystrophin protein and restoration of cardiomyocyte contractility and calcium transients to varying degrees. We show that deletion of exons 3-9 by genomic editing provides an especially effective means of correcting disease-causing ABD-1 mutations. These findings represent an important step toward eventual correction of common DMD mutations and provide a means of rapidly assessing the expression and function of internally truncated forms of dystrophin-lacking portions of ABD-1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/jci.insight.95918DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5621913PMC
September 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).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jacc.2017.06.061DOI Listing
August 2017

Defined Engineered Human Myocardium With Advanced Maturation for Applications in Heart Failure Modeling and Repair.

Circulation 2017 May 6;135(19):1832-1847. Epub 2017 Feb 6.

From Institute of Pharmacology and Toxicology, University Medical Center Göttingen, Germany (M.T., J.E.H., P.B., S.S., T.M., M.-L.C.L., E.L., F.R., S.Z., E. Wettwer, W.-H.Z.); German Center for Cardiovascular Research (DZHK), partner site Göttingen, Germany (M.T., J.E.H., P.B., S.S., T.M., M.-L.C.L., E.L., F.R., S.Z., E. Wingender, W.A.L., W.-H.Z.); Institute of Bioinformatics, University Medical Center Göttingen, Germany (S.Z., E. Wingender); Stanford Cardiovascular Institute (J.R., M.W., J.D.G., J.C.W.) and Department of Radiology (J.D.G., J.C.W.), Molecular Imaging Program, Stanford University School of Medicine, CA; The Sohnis Laboratory for Cardiac Electrophysiology and Regenerative Medicine, Technion-Israel Institute of Technology, Haifa (I.K., L.G.); Institute of Pharmacology and Toxicology, Technical University Dresden, Germany (E. Wettwer, U.R.); University Medical Center Utrecht and Hubrecht Institute, The Netherlands (P.D., L.W.v.L.); Leiden University Medical Center, The Netherlands (M.J.G.); Clinic for Cardiology and Pneumology, University Medical Center Göttingen, Germany (S.K., K.T., G.H., W.A.L.); Center for Applied Technology, Beckman Research Institute, City of Hope, Duarte, CA (L.A.C.); Department of Cardiovascular Physiology, Institute of Physiology, Ruhr University Bochum, Bochum, Germany (A.U., W.A.L.); New Laura and Isaac Perlmutter Cancer Center at New York University Langone (T.A., B.N.); and McEwen Centre for Regenerative Medicine, Toronto, Canada (G.K.). The current address for Dr Hudson is Laboratory for Cardiac Regeneration, School of Biomedical Sciences, The University of Queensland, Australia.

Background: Advancing structural and functional maturation of stem cell-derived cardiomyocytes remains a key challenge for applications in disease modeling, drug screening, and heart repair. Here, we sought to advance cardiomyocyte maturation in engineered human myocardium (EHM) toward an adult phenotype under defined conditions.

Methods: We systematically investigated cell composition, matrix, and media conditions to generate EHM from embryonic and induced pluripotent stem cell-derived cardiomyocytes and fibroblasts with organotypic functionality under serum-free conditions. We used morphological, functional, and transcriptome analyses to benchmark maturation of EHM.

Results: EHM demonstrated important structural and functional properties of postnatal myocardium, including: (1) rod-shaped cardiomyocytes with M bands assembled as a functional syncytium; (2) systolic twitch forces at a similar level as observed in bona fide postnatal myocardium; (3) a positive force-frequency response; (4) inotropic responses to β-adrenergic stimulation mediated via canonical β- and β-adrenoceptor signaling pathways; and (5) evidence for advanced molecular maturation by transcriptome profiling. EHM responded to chronic catecholamine toxicity with contractile dysfunction, cardiomyocyte hypertrophy, cardiomyocyte death, and N-terminal pro B-type natriuretic peptide release; all are classical hallmarks of heart failure. In addition, we demonstrate the scalability of EHM according to anticipated clinical demands for cardiac repair.

Conclusions: We provide proof-of-concept for a universally applicable technology for the engineering of macroscale human myocardium for disease modeling and heart repair from embryonic and induced pluripotent stem cell-derived cardiomyocytes under defined, serum-free conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCULATIONAHA.116.024145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5501412PMC
May 2017

Magnetic Resonance Imaging of Cardiac Strain Pattern Following Transplantation of Human Tissue Engineered Heart Muscles.

Circ Cardiovasc Imaging 2016 Nov;9(11)

From the Stanford Cardiovascular Institute and Department of Medicine, Division of Cardiology, CA (X.Q., J.R., X.Z., T.C., B.N., M.N., J.A., M.A., P.C.Y., P.N., J.C.W.); Auburn University MRI Research Center, Department of Electrical and Computer Engineering, AL (T.S.D.); Institute of Pharmacology, Heart Research Center, University Medical Center, Georg-August-University and German Center for Cardiovascular Research, Göttingen, Germany (M.T., W.H.Z.).

Background: The use of tissue engineering approaches in combination with exogenously produced cardiomyocytes offers the potential to restore contractile function after myocardial injury. However, current techniques assessing changes in global cardiac performance after such treatments are plagued by relatively low detection ability. Since the treatment is locally performed, this detection could be improved by myocardial strain imaging that measures regional contractility.

Methods And Results: Tissue engineered heart muscles (EHMs) were generated by casting human embryonic stem cell-derived cardiomyocytes with collagen in preformed molds. EHMs were transplanted (n=12) to cover infarct and border zones of recipient rat hearts 1 month after ischemia reperfusion injury. A control group (n=10) received only sham placement of sutures without EHMs. To assess the efficacy of EHMs, magnetic resonance imaging and ultrasound-based strain imaging were performed before and 4 weeks after transplantation. In addition to strain imaging, global cardiac performance was estimated from cardiac magnetic resonance imaging. Although no significant differences were found for global changes in left ventricular ejection fraction (control -9.6±1.3% versus EHM -6.2±1.9%; P=0.17), regional myocardial strain from tagged magnetic resonance imaging was able to detect preserved systolic function in EHM-treated animals compared with control (control 4.4±1.0% versus EHM 1.0±0.6%; P=0.04). However, ultrasound-based strain failed to detect any significant change (control 2.1±3.0% versus EHM 6.3±2.9%; P=0.46).

Conclusions: This study highlights the feasibility of using cardiac strain from tagged magnetic resonance imaging to assess functional changes in rat models following localized regenerative therapies, which may not be detected by conventional measures of global systolic performance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCIMAGING.116.004731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5378466PMC
November 2016

Hyperthermia Influences the Effects of Sodium Channel Blocking Drugs in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

PLoS One 2016 9;11(11):e0166143. Epub 2016 Nov 9.

First Department of Medicine, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany.

Introduction: Fever can increase the susceptibility to supraventricular and ventricular arrhythmias, in which sodium channel dysfunction has been implicated. Whether fever influences the efficacy of sodium channel blocking drugs is unknown. The current study was designed to investigate the temperature dependent effects of distinct sodium channel blocking drugs on the sodium currents in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs).

Methods And Results: hiPSC-CMs were generated from human skin fibroblasts of a healthy donor. The peak and late sodium currents (INa), steady-state activation, inactivation and recovery from inactivation of INa in hiPSC-CMs were analyzed using the whole-cell patch clamp technique. The effects of different concentrations of the antiarrhythmic drugs flecainide, lidocaine, ajmaline and the antianginal drug ranolazine on INa were tested at 36°C and 40°C. Increasing the temperature of the bath solution from 36°C to 40°C enhanced the inhibition of peak INa but reduced the inhibition of late INa by flecainide and lidocaine. By contrast, increasing the temperature reduced the effect of ajmaline and ranolazine on the peak INa but not late INa. None of the tested drugs showed temperature-dependent effects on the steady-state activation and inactivation as well as on the recovery from inactivation of INa in hiPSC-CMs.

Conclusions: Temperature variation from the physiological to the febrile range apparently influences the effects of sodium channel blockers on the sodium currents. This may influence their antiarrhythmic efficacy in patients suffering from fever.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0166143PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5102382PMC
June 2017

RhoA Ambivalently Controls Prominent Myofibroblast Characteritics by Involving Distinct Signaling Routes.

PLoS One 2015 8;10(10):e0137519. Epub 2015 Oct 8.

Institute of Pharmacology, University Medical Center Göttingen, Georg-August-University, Germany; DZHK (German Center for Cardiovascular Research), partner site Göttingen, Germany.

Introduction: RhoA has been shown to be beneficial in cardiac disease models when overexpressed in cardiomyocytes, whereas its role in cardiac fibroblasts (CF) is still poorly understood. During cardiac remodeling CF undergo a transition towards a myofibroblast phenotype thereby showing an increased proliferation and migration rate. Both processes involve the remodeling of the cytoskeleton. Since RhoA is known to be a major regulator of the cytoskeleton, we analyzed its role in CF and its effect on myofibroblast characteristics in 2 D and 3D models.

Results: Downregulation of RhoA was shown to strongly affect the actin cytoskeleton. It decreased the myofibroblast marker α-sm-actin, but increased certain fibrosis-associated factors like TGF-β and collagens. Also, the detailed analysis of CTGF expression demonstrated that the outcome of RhoA signaling strongly depends on the involved stimulus. Furthermore, we show that proliferation of myofibroblasts rely on RhoA and tubulin acetylation. In assays accessing three different types of migration, we demonstrate that RhoA/ROCK/Dia1 are important for 2D migration and the repression of RhoA and Dia1 signaling accelerates 3D migration. Finally, we show that a downregulation of RhoA in CF impacts the viscoelastic and contractile properties of engineered tissues.

Conclusion: RhoA positively and negatively influences myofibroblast characteristics by differential signaling cascades and depending on environmental conditions. These include gene expression, migration and proliferation. Reduction of RhoA leads to an increased viscoelasticity and a decrease in contractile force in engineered cardiac tissue.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0137519PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4598021PMC
June 2016

Evolutionarily conserved intercalated disc protein Tmem65 regulates cardiac conduction and connexin 43 function.

Nat Commun 2015 Sep 25;6:8391. Epub 2015 Sep 25.

Department of Physiology, University of Toronto, Toronto General Hospital Research Institute, Toronto, Ontario, Canada M5G 1L7.

Membrane proteins are crucial to heart function and development. Here we combine cationic silica-bead coating with shotgun proteomics to enrich for and identify plasma membrane-associated proteins from primary mouse neonatal and human fetal ventricular cardiomyocytes. We identify Tmem65 as a cardiac-enriched, intercalated disc protein that increases during development in both mouse and human hearts. Functional analysis of Tmem65 both in vitro using lentiviral shRNA-mediated knockdown in mouse cardiomyocytes and in vivo using morpholino-based knockdown in zebrafish show marked alterations in gap junction function and cardiac morphology. Molecular analyses suggest that Tmem65 interaction with connexin 43 (Cx43) is required for correct localization of Cx43 to the intercalated disc, since Tmem65 deletion results in marked internalization of Cx43, a shorter half-life through increased degradation, and loss of Cx43 function. Our data demonstrate that the membrane protein Tmem65 is an intercalated disc protein that interacts with and functionally regulates ventricular Cx43.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms9391DOI Listing
September 2015

p63RhoGEF regulates auto- and paracrine signaling in cardiac fibroblasts.

J Mol Cell Cardiol 2015 Nov 21;88:39-54. Epub 2015 Sep 21.

Institute of Pharmacology, Medical Center Goettingen, Germany; DZHK (German Center for Cardiovascular Research) partner sites Goettingen and Mannheim, Germany. Electronic address:

Cardiac remodeling, a hallmark of heart disease, is associated with intense auto- and paracrine signaling leading to cardiac fibrosis. We hypothesized that the specific mediator of Gq/11-dependent RhoA activation p63RhoGEF, which is expressed in cardiac fibroblasts, plays a role in the underlying processes. We could show that p63RhoGEF is up-regulated in mouse hearts subjected to transverse aortic constriction (TAC). In an engineered heart muscle model (EHM), p63RhoGEF expression in cardiac fibroblasts increased resting and twitch tensions, and the dominant negative p63ΔN decreased both. In an engineered connective tissue model (ECT), p63RhoGEF increased tissue stiffness and its knockdown as well as p63ΔN reduced stiffness. In 2D cultures of neonatal rat cardiac fibroblasts, p63RhoGEF regulated the angiotensin II (Ang II)-dependent RhoA activation, the activation of the serum response factor, and the expression and secretion of the connective tissue growth factor (CTGF). All these processes were inhibited by the knockdown of p63RhoGEF or by p63ΔN likely based on their negative influence on the actin cytoskeleton. Moreover, we show that p63RhoGEF also regulates CTGF in engineered tissues and correlates with it in the TAC model. Finally, confocal studies revealed a closely related localization of p63RhoGEF and CTGF in the trans-Golgi network.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.yjmcc.2015.09.009DOI Listing
November 2015

Human Engineered Heart Muscles Engraft and Survive Long Term in a Rodent Myocardial Infarction Model.

Circ Res 2015 Sep 19;117(8):720-30. Epub 2015 Aug 19.

From the Division of Cardiology, Department of Medicine, Stanford Cardiovascular Institute (J.R., A.E., E.T., U.R., O.J.A., O.S., N.G.K., E.N., M.W., P.S.T., J.D.G., J.C.W.) and Department of Pathology (A.J.C.), Stanford University School of Medicine, CA; Department for Research and Development, Veterans Administration Palo Alto Health Care System, CA (P.S.T.); Institute of Pharmacology, Heart Research Center, University Medical Center, Georg-August-University and German Center for Cardiovascular Research, Göttingen, Germany (M.T., T.M., W.H.Z.); and Center for Biomedicine and Genetics (V.C.C., L.A.C.) and Center for Applied Technology Development, Beckman Research Institute (A.J.C.), City of Hope, Duarte, CA.

Rationale: Tissue engineering approaches may improve survival and functional benefits from human embryonic stem cell-derived cardiomyocyte transplantation, thereby potentially preventing dilative remodeling and progression to heart failure.

Objective: Assessment of transport stability, long-term survival, structural organization, functional benefits, and teratoma risk of engineered heart muscle (EHM) in a chronic myocardial infarction model.

Methods And Results: We constructed EHMs from human embryonic stem cell-derived cardiomyocytes and released them for transatlantic shipping following predefined quality control criteria. Two days of shipment did not lead to adverse effects on cell viability or contractile performance of EHMs (n=3, P=0.83, P=0.87). One month after ischemia/reperfusion injury, EHMs were implanted onto immunocompromised rat hearts to simulate chronic ischemia. Bioluminescence imaging showed stable engraftment with no significant cell loss between week 2 and 12 (n=6, P=0.67), preserving ≤25% of the transplanted cells. Despite high engraftment rates and attenuated disease progression (change in ejection fraction for EHMs, -6.7±1.4% versus control, -10.9±1.5%; n>12; P=0.05), we observed no difference between EHMs containing viable and nonviable human cardiomyocytes in this chronic xenotransplantation model (n>12; P=0.41). Grafted cardiomyocytes showed enhanced sarcomere alignment and increased connexin 43 expression at 220 days after transplantation. No teratomas or tumors were found in any of the animals (n=14) used for long-term monitoring.

Conclusions: EHM transplantation led to high engraftment rates, long-term survival, and progressive maturation of human cardiomyocytes. However, cell engraftment was not correlated with functional improvements in this chronic myocardial infarction model. Most importantly, the safety of this approach was demonstrated by the lack of tumor or teratoma formation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCRESAHA.115.306985DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4679370PMC
September 2015

Physiologic force-frequency response in engineered heart muscle by electromechanical stimulation.

Biomaterials 2015 Aug 15;60:82-91. Epub 2015 May 15.

Institute of Pharmacology, Heart Research Center Göttingen, University Medical Center Göttingen, Georg-August-University Göttingen, 37075, Germany; German Center for Cardiovascular Research (DZHK), Partner Site Göttingen, 37075, Germany. Electronic address:

A hallmark of mature mammalian ventricular myocardium is a positive force-frequency relationship (FFR). Despite evidence of organotypic structural and molecular maturation, a positive FFR has not been observed in mammalian tissue engineered heart muscle. We hypothesized that concurrent mechanical and electrical stimulation at frequencies matching physiological heart rate will result in functional maturation. We investigated the role of biomimetic mechanical and electrical stimulation in functional maturation in engineered heart muscle (EHM). Following tissue consolidation, EHM were subjected to electrical field stimulation at 0, 2, 4, or 6 Hz for 5 days, while strained on flexible poles to facilitate auxotonic contractions. EHM stimulated at 2 and 4 Hz displayed a similarly enhanced inotropic reserve, but a clearly diverging FFR. The positive FFR in 4 Hz stimulated EHM was associated with reduced calcium sensitivity, frequency-dependent acceleration of relaxation, and enhanced post-rest potentiation. This was paralleled on the cellular level with improved calcium storage and release capacity of the sarcoplasmic reticulum and enhanced T-tubulation. We conclude that electro-mechanical stimulation at a physiological frequency supports functional maturation in mammalian EHM. The observed positive FFR in EHM has important implications for the applicability of EHM in cardiovascular research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biomaterials.2015.03.055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4921199PMC
August 2015

Pre- and post-conditional inhibition of prolyl-4-hydroxylase domain enzymes protects the heart from an ischemic insult.

Pflugers Arch 2015 Oct 13;467(10):2141-9. Epub 2015 Jan 13.

Institute of Cardiovascular Physiology, University Medical Center, Georg-August-University Göttingen, Humboldtallee 23, 37073, Göttingen, Germany.

Several genetically modified mouse models implicated that prolyl-4-hydroxylase domain (PHD) enzymes are critical mediators for protecting tissues from an ischemic insult including myocardial infarction by affecting the stability and activation of hypoxia-inducible factor (HIF)-1 and HIF-2. Thus, the current efforts to develop small-molecule PHD inhibitors open a new therapeutic option for myocardial tissue protection during ischemia. Therefore, we aimed to investigate the applicability and efficacy of pharmacological HIFα stabilization by a small-molecule PHD inhibitor in the heart. We tested for protective effects in the acute phase of myocardial infarction after pre- or post-conditional application of the inhibitor. Application of the specific PHD inhibitor 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate (ICA) resulted in HIF-1α and HIF-2α accumulation in heart muscle cells in vitro and in vivo. The rapid and robust responsiveness of cardiac tissue towards ICA was further confirmed by induction of the known HIF target genes heme oxygenase-1 and PHD3. Pre- and post-conditional treatment of mice undergoing myocardial infarction resulted in a significantly smaller infarct size. Tissue protection from ischemia after pre- or post-conditional ICA treatment demonstrates that there is a therapeutic time window for the application of the PHD inhibitor (PHI) post-myocardial infarction, which might be exploited for acute medical interventions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00424-014-1667-zDOI Listing
October 2015