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The fluorescence ubiquitination cell cycle inhibitor (FUCCI) has been introduced to monitor cell cycle activity in living cells, including human induced pluripotent stem cells (hiPSC) and derived cell types. We have recently developed hiPSC with stable expression of dCas9VPR for endogenous gene activation and a Citrine-tagged ACTN2 cell line to monitor sarcomere development and function in muscle cells. Here, we present dual and triple transgenic hiPSC lines developed by genomic integration of FUCCI with and without dCas9VPR into the ROSA26 and AAVS1 loci, respectively, in the previously introduced ACTN2-Citrine line. Functionality of the transgenes was demonstrated in the novel hiPSC line, which we introduce as Myo-CCER and CraCCER.
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Ciclo Celular , Células Madre Pluripotentes Inducidas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Línea CelularRESUMEN
AIMS: Atrial fibrillation (AF) is associated with tachycardia-induced cellular electrophysiology alterations which promote AF chronification and treatment resistance. Development of novel antiarrhythmic therapies is hampered by the absence of scalable experimental human models that reflect AF-associated electrical remodelling. Therefore, we aimed to assess if AF-associated remodelling of cellular electrophysiology can be simulated in human atrial-like cardiomyocytes derived from induced pluripotent stem cells in the presence of retinoic acid (iPSC-aCM), and atrial-engineered human myocardium (aEHM) under short term (24 h) and chronic (7 days) tachypacing (TP). METHODS AND RESULTS: First, 24-h electrical pacing at 3 Hz was used to investigate whether AF-associated remodelling in iPSC-aCM and aEHM would ensue. Compared to controls (24 h, 1 Hz pacing) TP-stimulated iPSC-aCM presented classical hallmarks of AF-associated remodelling: (i) decreased L-type Ca2+ current (ICa,L) and (ii) impaired activation of acetylcholine-activated inward-rectifier K+ current (IK,ACh). This resulted in action potential shortening and an absent response to the M-receptor agonist carbachol in both iPSC-aCM and aEHM subjected to TP. Accordingly, mRNA expression of the channel-subunit Kir3.4 was reduced. Selective IK,ACh blockade with tertiapin reduced basal inward-rectifier K+ current only in iPSC-aCM subjected to TP, thereby unmasking an agonist-independent constitutively active IK,ACh. To allow for long-term TP, we developed iPSC-aCM and aEHM expressing the light-gated ion-channel f-Chrimson. The same hallmarks of AF-associated remodelling were observed after optical-TP. In addition, continuous TP (7 days) led to (i) increased amplitude of inward-rectifier K+ current (IK1), (ii) hyperpolarization of the resting membrane potential, (iii) increased action potential-amplitude and upstroke velocity as well as (iv) reversibly impaired contractile function in aEHM. CONCLUSIONS: Classical hallmarks of AF-associated remodelling were mimicked through TP of iPSC-aCM and aEHM. The use of the ultrafast f-Chrimson depolarizing ion channel allowed us to model the time-dependence of AF-associated remodelling in vitro for the first time. The observation of electrical remodelling with associated reversible contractile dysfunction offers a novel platform for human-centric discovery of antiarrhythmic therapies.
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Fibrilación Atrial , Remodelación Atrial , Células Madre Pluripotentes Inducidas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/metabolismo , Atrios Cardíacos , Antiarrítmicos/farmacología , Antiarrítmicos/uso terapéutico , Potenciales de Acción , Acetilcolina/farmacologíaRESUMEN
Arrhythmogenic cardiomyopathy is a severe cardiac disorder characterized by lethal arrhythmias and sudden cardiac death, with currently no effective treatment. Plakophilin 2 (PKP2) is the most frequently affected gene. Here we show that adeno-associated virus (AAV)-mediated delivery of PKP2 in PKP2c.2013delC/WT induced pluripotent stem cell-derived cardiomyocytes restored not only cardiac PKP2 levels but also the levels of other junctional proteins, found to be decreased in response to the mutation. PKP2 restoration improved sodium conduction, indicating rescue of the arrhythmic substrate in PKP2 mutant induced pluripotent stem cell-derived cardiomyocytes. Additionally, it enhanced contractile function and normalized contraction kinetics in PKP2 mutant engineered human myocardium. Recovery of desmosomal integrity and cardiac function was corroborated in vivo, by treating heterozygous Pkp2c.1755delA knock-in mice. Long-term treatment with AAV9-PKP2 prevented cardiac dysfunction in 12-month-old Pkp2c.1755delA/WT mice, without affecting wild-type mice. These findings encourage clinical exploration of PKP2 gene therapy for patients with PKP2 haploinsufficiency.
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Heterozygous truncating variants in TTN (TTNtv), the gene coding for titin, cause dilated cardiomyopathy (DCM), but the underlying pathomechanisms are unclear and disease management remains uncertain. Truncated titin proteins have not yet been considered as a contributor to disease development. Here, we studied myocardial tissues from nonfailing donor hearts and 113 patients with end-stage DCM for titin expression and identified a TTNtv in 22 patients with DCM (19.5%). We directly demonstrate titin haploinsufficiency in TTNtv-DCM hearts and the absence of compensatory changes in the alternative titin isoform Cronos. Twenty-one TTNtv-DCM hearts in our cohort showed stable expression of truncated titin proteins. Expression was variable, up to half of the total titin protein pool, and negatively correlated with patient age at heart transplantation. Truncated titin proteins were not detected in sarcomeres but were present in intracellular aggregates, with deregulated ubiquitin-dependent protein quality control. We produced human induced pluripotent stem cellderived cardiomyocytes (hiPSC-CMs), comparing wild-type controls to cells with a patient-derived, prototypical A-band-TTNtv or a CRISPR-Cas9generated M-band-TTNtv. TTNtv-hiPSC-CMs showed reduced wild-type titin expression and contained truncated titin proteins whose proportion increased upon inhibition of proteasomal activity. In engineered heart muscle generated from hiPSC-CMs, depressed contractility caused by TTNtv could be reversed by correction of the mutation using CRISPR-Cas9, eliminating truncated titin proteins and raising wild-type titin content. Functional improvement also occurred when wild-type titin protein content was increased by proteasome inhibition. Our findings reveal the major pathomechanisms of TTNtv-DCM and can be exploited for new therapies to treat TTNtv-related cardiomyopathies.
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Cardiomiopatías , Conectina , Trasplante de Corazón , Células Madre Pluripotentes Inducidas , Cardiomiopatías/genética , Conectina/genética , Conectina/metabolismo , Haploinsuficiencia , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Mutación , Miocitos Cardíacos/metabolismo , Donantes de TejidosRESUMEN
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 AQP1 is a major isoform. AQP1 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 (H2O2), produced extracellularly, is necessary for the hypertrophic response of isolated cardiac myocytes and that AQP1 facilitates the transmembrane transport of H2O2 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 H2O2 Deletion of Aqp1 or selective blockade of the AQP1 intrasubunit pore inhibited H2O2 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 H2O2 by the water channel AQP1 and that inhibitors of AQP1 represent new possibilities for treating hypertrophic cardiomyopathies.
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Acuaporina 1 , Células Madre Pluripotentes Inducidas , Animales , Humanos , Peróxido de Hidrógeno/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Miocardio/metabolismo , Miocitos Cardíacos/metabolismoRESUMEN
RATIONALE: Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is evolving rapidly. Recently, second-generation CRISPR/Cas9 activation systems based on nuclease inactive dead (d)Cas9 fused to transcriptional transactivation domains were developed for directing specific guide (g)RNAs to regulatory regions of any gene of interest, to enhance transcription. The application of dCas9 to activate cardiomyocyte transcription in targeted genomic loci in vivo has not been demonstrated so far. OBJECTIVE: We aimed to develop a mouse model for cardiomyocyte-specific, CRISPR-mediated transcriptional modulation, and to demonstrate its versatility by targeting Mef2d and Klf15 loci (2 well-characterized genes implicated in cardiac hypertrophy and homeostasis) for enhanced transcription. METHODS AND RESULTS: A mouse model expressing dCas9 with the VPR transcriptional transactivation domains under the control of the Myh (myosin heavy chain) 6 promoter was generated. These mice innocuously expressed dCas9 exclusively in cardiomyocytes. For initial proof-of-concept, we selected Mef2d, which when overexpressed, led to hypertrophy and heart failure, and Klf15, which is lowly expressed in the neonatal heart. The most effective gRNAs were first identified in fibroblast (C3H/10T1/2) and myoblast (C2C12) cell lines. Using an improved triple gRNA expression system (TRISPR [triple gRNA expression construct]), up to 3 different gRNAs were transduced simultaneously to identify optimal conditions for transcriptional activation. For in vivo delivery of the validated gRNA combinations, we employed systemic administration via adeno-associated virus serotype 9. On gRNA delivery targeting Mef2d expression, we recapitulated the anticipated cardiac hypertrophy phenotype. Using gRNA targeting Klf15, we could enhance its transcription significantly, although Klf15 is physiologically silenced at that time point. We further confirmed specific and robust dCas9VPR on-target effects. CONCLUSIONS: The developed mouse model permits enhancement of gene expression by using endogenous regulatory genomic elements. Proof-of-concept in 2 independent genomic loci suggests versatile applications in controlling transcription in cardiomyocytes of the postnatal heart.
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Sistemas CRISPR-Cas , Regulación de la Expresión Génica , Miocardio/metabolismo , Activación Transcripcional , Animales , Línea Celular , Dependovirus/genética , Fibroblastos/metabolismo , Regulación de la Expresión Génica/genética , Genes Sintéticos , Vectores Genéticos/genética , Corazón/crecimiento & desarrollo , Factores de Transcripción de Tipo Kruppel/biosíntesis , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción MEF2/biosíntesis , Factores de Transcripción MEF2/genética , Ratones , Ratones Transgénicos , Miocitos Cardíacos/metabolismo , Cadenas Pesadas de Miosina/genética , Regiones Promotoras Genéticas , Dominios Proteicos , ARN Polimerasa III/genética , ARN Guía de Kinetoplastida/genéticaRESUMEN
Deterioration or inborn malformations of the cardiac conduction system (CCS) interfere with proper impulse propagation in the heart and may lead to sudden cardiac death or heart failure. Patients afflicted with arrhythmia depend on antiarrhythmic medication or invasive therapy, such as pacemaker implantation. An ideal way to treat these patients would be CCS tissue restoration. This, however, requires precise knowledge regarding the molecular mechanisms underlying CCS development. Here, we aimed to identify regulators of CCS development. We performed a compound screen in zebrafish embryos and identified tolterodine, a muscarinic receptor antagonist, as a modifier of CCS development. Tolterodine provoked a lower heart rate, pericardiac edema, and arrhythmia. Blockade of muscarinic M3, but not M2, receptors induced transcriptional changes leading to amplification of sinoatrial cells and loss of atrioventricular identity. Transcriptome data from an engineered human heart muscle model provided additional evidence for the contribution of muscarinic M3 receptors during cardiac progenitor specification and differentiation. Taken together, we found that muscarinic M3 receptors control the CCS already before the heart becomes innervated. Our data indicate that muscarinic receptors maintain a delicate balance between the developing sinoatrial node and the atrioventricular canal, which is probably required to prevent the development of arrhythmia.
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Arritmias Cardíacas/tratamiento farmacológico , Sistema de Conducción Cardíaco/embriología , Antagonistas Muscarínicos/farmacología , Organogénesis/efectos de los fármacos , Receptor Muscarínico M3/metabolismo , Tartrato de Tolterodina/farmacología , Animales , Arritmias Cardíacas/fisiopatología , Modelos Animales de Enfermedad , Embrión de Mamíferos , Embrión no Mamífero , Células HEK293 , Sistema de Conducción Cardíaco/efectos de los fármacos , Sistema de Conducción Cardíaco/fisiopatología , Humanos , Ratones , Ratones Noqueados , Antagonistas Muscarínicos/uso terapéutico , Miocitos Cardíacos , Receptor Muscarínico M3/genética , Tartrato de Tolterodina/uso terapéutico , Xenopus laevis , Pez CebraRESUMEN
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).
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Arritmias Cardíacas/tratamiento farmacológico , Evaluación Preclínica de Medicamentos , Matriz Extracelular/metabolismo , Corazón/fisiología , Células Madre Pluripotentes Inducidas/citología , Modelos Cardiovasculares , Miocitos Cardíacos/citología , Ingeniería de Tejidos/métodos , Potenciales de Acción/efectos de los fármacos , Animales , Arritmias Cardíacas/patología , Calcio/metabolismo , Fármacos Cardiovasculares/farmacología , Modelos Animales de Enfermedad , Matriz Extracelular/efectos de los fármacos , Humanos , Hidrogeles/farmacología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Especificidad de Órganos , PorcinosRESUMEN
The sympathetic nervous system is the main stimulator of cardiac function. While acute activation of the ß-adrenoceptors exerts positive inotropic and lusitropic effects by increasing cAMP and Ca2+, chronically enhanced sympathetic tone with changed ß-adrenergic signaling leads to alterations of gene expression and remodeling. The CREB-regulated transcription coactivator 1 (CRTC1) is activated by cAMP and Ca2+. In the present study, the regulation of CRTC1 in cardiomyocytes and its effect on cardiac function and growth was investigated. In cardiomyocytes, isoprenaline induced dephosphorylation, and thus activation of CRTC1, which was prevented by propranolol. Crtc1-deficient mice exhibited left ventricular dysfunction, hypertrophy and enlarged cardiomyocytes. However, isoprenaline-induced contractility of isolated trabeculae or phosphorylation of cardiac troponin I, cardiac myosin-binding protein C, phospholamban, and ryanodine receptor were not altered, suggesting that cardiac dysfunction was due to the global lack of Crtc1. The mRNA and protein levels of the Gαq GTPase activating protein regulator of G-protein signaling 2 (RGS2) were lower in hearts of Crtc1-deficient mice. Chromatin immunoprecipitation and reporter gene assays showed stimulation of the Rgs2 promoter by CRTC1. In Crtc1-deficient cardiomyocytes, phosphorylation of the Gαq-downstream kinase ERK was enhanced. CRTC1 content was higher in cardiac tissue from patients with aortic stenosis or hypertrophic cardiomyopathy and from two murine models mimicking these diseases. These data suggest that increased CRTC1 in maladaptive hypertrophy presents a compensatory mechanism to delay disease progression in part by enhancing Rgs2 gene transcription. Furthermore, the present study demonstrates an important role of CRTC1 in the regulation of cardiac function and growth.
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Cardiomegalia/metabolismo , Factores de Transcripción/metabolismo , Animales , Cardiomegalia/diagnóstico por imagen , Cardiomegalia/fisiopatología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células HEK293 , Humanos , Ratones Endogámicos C57BL , Miocitos Cardíacos/metabolismo , Fosforilación , Regiones Promotoras Genéticas , Proteínas RGS/genética , Proteínas RGS/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Wistar , Receptores Adrenérgicos beta/metabolismo , Transducción de Señal , Factores de Transcripción/deficienciaRESUMEN
Acute heart failure (HF) and in particular, cardiogenic shock are associated with high morbidity and mortality. A therapeutic dilemma is that the use of positive inotropic agents, such as catecholamines or phosphodiesterase-inhibitors, is associated with increased mortality. Newer drugs, such as levosimendan or omecamtiv mecarbil, target sarcomeres to improve systolic function putatively without elevating intracellular Ca2+. Although meta-analyses of smaller trials suggested that levosimendan is associated with a better outcome than dobutamine, larger comparative trials failed to confirm this observation. For omecamtiv mecarbil, Phase II clinical trials suggest a favourable haemodynamic profile in patients with acute and chronic HF, and a Phase III morbidity/mortality trial in patients with chronic HF has recently begun. Here, we review the pathophysiological basis of systolic dysfunction in patients with HF and the mechanisms through which different inotropic agents improve cardiac function. Since adenosine triphosphate and reactive oxygen species production in mitochondria are intimately linked to the processes of excitation-contraction coupling, we also discuss the impact of inotropic agents on mitochondrial bioenergetics and redox regulation. Therefore, this position paper should help identify novel targets for treatments that could not only safely improve systolic and diastolic function acutely, but potentially also myocardial structure and function over a longer-term.
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Cardiotónicos/uso terapéutico , Acoplamiento Excitación-Contracción/efectos de los fármacos , Insuficiencia Cardíaca/tratamiento farmacológico , Choque Cardiogénico/tratamiento farmacológico , Enfermedad Aguda , Animales , Antioxidantes/efectos adversos , Antioxidantes/uso terapéutico , Calcio/metabolismo , Cardiotónicos/efectos adversos , Estudios de Casos y Controles , Catecolaminas/efectos adversos , Catecolaminas/uso terapéutico , Ensayos Clínicos como Asunto , Diástole/efectos de los fármacos , Dobutamina/efectos adversos , Dobutamina/uso terapéutico , Perros , Metabolismo Energético/efectos de los fármacos , Insuficiencia Cardíaca/mortalidad , Humanos , Mitocondrias/metabolismo , Modelos Animales , Contracción Miocárdica/efectos de los fármacos , Óxidos de Nitrógeno/efectos adversos , Óxidos de Nitrógeno/uso terapéutico , Oxidación-Reducción/efectos de los fármacos , Inhibidores de Fosfodiesterasa/efectos adversos , Inhibidores de Fosfodiesterasa/uso terapéutico , Placebos/administración & dosificación , Receptores Adrenérgicos/efectos de los fármacos , Sarcómeros/efectos de los fármacos , Sarcómeros/metabolismo , Choque Cardiogénico/mortalidad , Simendán/efectos adversos , Simendán/uso terapéutico , Porcinos , Sístole/efectos de los fármacos , Urea/efectos adversos , Urea/análogos & derivados , Urea/uso terapéuticoRESUMEN
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 × 106 hPSC-CMs were mixed with 0.4 × 106 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, ß-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.
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Biología Computacional/métodos , Miocardio/citología , Línea Celular , Citometría de Flujo , Humanos , Miocardio/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Ingeniería de Tejidos/métodosRESUMEN
Our current understanding of cardiac excitation and its coupling to contraction is largely based on ex vivo studies utilising fluorescent organic dyes to assess cardiac action potentials and signal transduction. Recent advances in optogenetic sensors open exciting new possibilities for cardiac research and allow us to answer research questions that cannot be addressed using the classic organic dyes. Especially thrilling is the possibility to use optogenetic sensors to record parameters of cardiac excitation and contraction in vivo. In addition, optogenetics provide a high spatial resolution, as sensors can be coupled to motifs and targeted to specific cell types and subcellular domains of the heart. In this review, we will give a comprehensive overview of relevant optogenetic sensors, how they can be utilised in cardiac research and how they have been applied in cardiac research up to now.
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Investigación Biomédica/métodos , Técnicas Biosensibles , Señalización del Calcio , Cardiología/métodos , Corazón/fisiología , Canales Iónicos/metabolismo , Contracción Miocárdica , Miocitos Cardíacos/metabolismo , Optogenética , Potenciales de Acción , Animales , Acoplamiento Excitación-Contracción , Humanos , Transporte IónicoRESUMEN
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.
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Diferenciación Celular , Células Madre Embrionarias Humanas/trasplante , Imagen por Resonancia Cinemagnética , Contracción Miocárdica , Infarto del Miocardio/cirugía , Miocardio/patología , Miocitos Cardíacos/trasplante , Regeneración , Ingeniería de Tejidos/métodos , Andamios del Tejido , Animales , Fenómenos Biomecánicos , Línea Celular , Modelos Animales de Enfermedad , Ecocardiografía , Estudios de Factibilidad , Xenoinjertos , Humanos , Masculino , Infarto del Miocardio/diagnóstico por imagen , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Miocitos Cardíacos/patología , Fenotipo , Valor Predictivo de las Pruebas , Ratas Desnudas , Recuperación de la Función , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
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.
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Conexina 43/metabolismo , Uniones Comunicantes/metabolismo , Sistema de Conducción Cardíaco/metabolismo , Ventrículos Cardíacos/metabolismo , Proteínas de la Membrana/genética , Miocitos Cardíacos/metabolismo , Proteínas de Pez Cebra/genética , Animales , Western Blotting , Cromatografía Líquida de Alta Presión , Técnica del Anticuerpo Fluorescente , Uniones Comunicantes/ultraestructura , Técnicas de Silenciamiento del Gen , Sistema de Conducción Cardíaco/fisiología , Técnicas In Vitro , Proteínas de la Membrana/metabolismo , Ratones , Microscopía Electrónica , Miocitos Cardíacos/fisiología , Miocitos Cardíacos/ultraestructura , Proteómica , Dióxido de Silicio , Pez Cebra , Proteínas de Pez Cebra/metabolismoRESUMEN
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.
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Células Madre Embrionarias/trasplante , Supervivencia de Injerto , Trasplante de Corazón/métodos , Infarto del Miocardio/cirugía , Miocitos Cardíacos/trasplante , Músculos Papilares/trasplante , Ingeniería de Tejidos/métodos , Animales , Biomarcadores/metabolismo , Diferenciación Celular , Línea Celular , Supervivencia Celular , Conexina 43/metabolismo , Modelos Animales de Enfermedad , Células Madre Embrionarias/inmunología , Células Madre Embrionarias/metabolismo , Trasplante de Corazón/efectos adversos , Xenoinjertos , Humanos , Inmunosupresores/farmacología , Masculino , Contracción Miocárdica , Infarto del Miocardio/inmunología , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Miocitos Cardíacos/inmunología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Músculos Papilares/inmunología , Músculos Papilares/metabolismo , Músculos Papilares/patología , Músculos Papilares/fisiopatología , Ratas Desnudas , Ratas Sprague-Dawley , Volumen Sistólico , Factores de Tiempo , TransfecciónRESUMEN
Restoring expression levels of the EF-hand calcium (Ca(2+)) sensor protein S100A1 has emerged as a key factor in reconstituting normal Ca(2+) handling in failing myocardium. Improved sarcoplasmic reticulum (SR) function with enhanced Ca(2+) resequestration appears critical for S100A1's cyclic adenosine monophosphate-independent inotropic effects but raises concerns about potential diastolic SR Ca(2+) leakage that might trigger fatal arrhythmias. This study shows for the first time a diminished interaction between S100A1 and ryanodine receptors (RyR2s) in experimental HF. Restoring this link in failing cardiomyocytes, engineered heart tissue and mouse hearts, respectively, by means of adenoviral and adeno-associated viral S100A1 cDNA delivery normalizes diastolic RyR2 function and protects against Ca(2+)- and ß-adrenergic receptor-triggered proarrhythmogenic SR Ca(2+) leakage in vitro and in vivo. S100A1 inhibits diastolic SR Ca(2+) leakage despite aberrant RyR2 phosphorylation via protein kinase A and calmodulin-dependent kinase II and stoichiometry with accessory modulators such as calmodulin, FKBP12.6 or sorcin. Our findings demonstrate that S100A1 is a regulator of diastolic RyR2 activity and beneficially modulates diastolic RyR2 dysfunction. S100A1 interaction with the RyR2 is sufficient to protect against basal and catecholamine-triggered arrhythmic SR Ca(2+) leak in HF, combining antiarrhythmic potency with chronic inotropic actions.
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Insuficiencia Cardíaca/genética , Canal Liberador de Calcio Receptor de Rianodina/genética , Proteínas S100/metabolismo , Animales , Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Calmodulina/metabolismo , ADN Complementario/metabolismo , Electrocardiografía , Técnicas de Transferencia de Gen , Insuficiencia Cardíaca/prevención & control , Masculino , Ratones , Microscopía Fluorescente , Miocardio/metabolismo , Miocitos Cardíacos/citología , Fosforilación , Unión Proteica , Ratas , Ratas Sprague-Dawley , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Proteínas de Unión a Tacrolimus/metabolismo , Ingeniería de Tejidos/métodosRESUMEN
miR-133a and miR-1 are known as muscle-specific microRNAs that are involved in cardiac development and pathophysiology. We have shown that both miR-1 and miR-133a are early and progressively upregulated during in vitro cardiac differentiation of adult cardiac progenitor cells (CPCs), but only miR-133a expression was enhanced under in vitro oxidative stress. miR-1 was demonstrated to favor differentiation of CPCs, whereas miR-133a overexpression protected CPCs against cell death, targeting, among others, the proapoptotic genes Bim and Bmf. miR-133a-CPCs clearly improved cardiac function in a rat myocardial infarction model by reducing fibrosis and hypertrophy and increasing vascularization and cardiomyocyte proliferation. The beneficial effects of miR-133a-CPCs seem to correlate with the upregulated expression of several relevant paracrine factors and the plausible cooperative secretion of miR-133a via exosomal transport. Finally, an in vitro heart muscle model confirmed the antiapoptotic effects of miR-133a-CPCs, favoring the structuration and contractile functionality of the artificial tissue.
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MicroARNs/genética , Mioblastos Cardíacos/metabolismo , Infarto del Miocardio/genética , Animales , Cardiomegalia/genética , Cardiomegalia/patología , Biología Computacional , Expresión Génica , Perfilación de la Expresión Génica , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Interferencia de ARN , ARN Mensajero/genética , RatasRESUMEN
BACKGROUND/AIMS: The hypoxia inducible factor-1 (HIF-1) is a suitable marker for tissue oxygenation. We intended to develop cardiomyocytes (CMs) expressing the oxygen-dependent degradation domain of HIF-1α fused to the firefly luciferase (ODD-Luc) followed by proof-of-concept for its applicability in the assessment of heart muscle oxygenation. METHODS AND RESULTS: We first generated embryonic stem cell (ESC) lines (ODD-Luc ESCs) from a Tg ROSA26 ODD-Luc/+ mouse. Subsequent CMs selection was facilitated by stable integration of an antibiotic resistance expressed under the control of the αMHC promoter. ODD-Luc ESCs showed a strong Luc-signal within 1 h of hypoxia (1% oxygen), which coincided with endogenous HIF-1α. Engineered heart muscle (EHM) constructed with ODD-Luc CMs confirmed the utility of the model to sense hypoxia, and monitor reoxygenation also in a multicellular heart muscle model. Pharmacologically induced inotropy/chronotropy under isoprenaline resulted in enhanced Luc-signal suggesting enhanced oxygen consumption, leading to notable myocardial hypoxia. CONCLUSIONS: ODD-Luc-CMs can be used to monitor dynamic changes of cardiomyocyte oxygenation in living heart muscle samples. We provide proof-of-concept for pharmacologically induced myocardial interventions and envision applications of the developed model in drug screens and fundamental studies of ischemia/reperfusion injury.
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Ingeniería Genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Miocitos Cardíacos/metabolismo , Animales , RatonesRESUMEN
AIMS: Induced pluripotent stem cells (iPSCs) provide a unique opportunity for the generation of patient-specific cells for use in disease modelling, drug screening, and regenerative medicine. The aim of this study was to compare human-induced pluripotent stem cells (hiPSCs) derived from different somatic cell sources regarding their generation efficiency and cardiac differentiation potential, and functionalities of cardiomyocytes. METHODS AND RESULTS: We generated hiPSCs from hair keratinocytes, bone marrow mesenchymal stem cells (MSCs), and skin fibroblasts by using two different virus systems. We show that MSCs and fibroblasts are more easily reprogrammed than keratinocytes. This corresponds to higher methylation levels of minimal promoter regions of the OCT4 and NANOG genes in keratinocytes than in MSCs and fibroblasts. The success rate and reprogramming efficiency was significantly higher by using the STEMCCA system than the OSNL system. All analysed hiPSCs are pluripotent and show phenotypical characteristics similar to human embryonic stem cells. We studied the cardiac differentiation efficiency of generated hiPSC lines (n = 24) and found that MSC-derived hiPSCs exhibited a significantly higher efficiency to spontaneously differentiate into beating cardiomyocytes when compared with keratinocyte-, and fibroblast-derived hiPSCs. There was no significant difference in the functionalities of the cardiomyocytes derived from hiPSCs with different origins, showing the presence of pacemaker-, atrial-, ventricular- and Purkinje-like cardiomyocytes, and exhibiting rhythmic Ca2+ transients and Ca2+ sparks in hiPSC-derived cardiomyocytes. Furthermore, spontaneously and synchronously beating and force-developing engineered heart tissues were generated. CONCLUSIONS: Human-induced pluripotent stem cells can be reprogrammed from all three somatic cell types, but with different efficiency. All analysed iPSCs can differentiate into cardiomyocytes, and the functionalities of cardiomyocytes derived from different cell origins are similar. However, MSC-derived hiPSCs revealed a higher cardiac differentiation efficiency than keratinocyte- and fibroblast-derived hiPSCs.
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Células de la Médula Ósea/citología , Fibroblastos/citología , Cabello/citología , Células Madre Pluripotentes Inducidas/citología , Queratinocitos/citología , Piel/citología , Potenciales de Acción/fisiología , Biomarcadores/metabolismo , Calcio/metabolismo , Diferenciación Celular/fisiología , Células Cultivadas , Reprogramación Celular/fisiología , Metilación de ADN/fisiología , Epigénesis Genética , Proteínas de Homeodominio/metabolismo , Humanos , Células Madre Mesenquimatosas/citología , Proteína Homeótica Nanog , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factores de Transcripción SOXB1/metabolismo , Ingeniería de TejidosRESUMEN
BACKGROUND: The angiotensin II receptor subtype 2 (AT2 receptor) is ubiquitously and highly expressed in early postnatal life. However, its role in postnatal cardiac development remained unclear. METHODOLOGY/PRINCIPAL FINDINGS: Hearts from 1, 7, 14 and 56 days old wild-type (WT) and AT2 receptor-deficient (KO) mice were extracted for histomorphometrical analysis as well as analysis of cardiac signaling and gene expression. Furthermore, heart and body weights of examined animals were recorded and echocardiographic analysis of cardiac function as well as telemetric blood pressure measurements were performed. Moreover, gene expression, sarcomere shortening and calcium transients were examined in ventricular cardiomyocytes isolated from both genotypes. KO mice exhibited an accelerated body weight gain and a reduced heart to body weight ratio as compared to WT mice in the postnatal period. However, in adult KO mice the heart to body weight ratio was significantly increased most likely due to elevated systemic blood pressure. At postnatal day 7 ventricular capillarization index and the density of α-smooth muscle cell actin-positive blood vessels were higher in KO mice as compared to WT mice but normalized during adolescence. Echocardiographic assessment of cardiac systolic function at postnatal day 7 revealed decreased contractility of KO hearts in response to beta-adrenergic stimulation. Moreover, cardiomyocytes from KO mice showed a decreased sarcomere shortening and an increased peak Ca(2+) transient in response to isoprenaline when stimulated concomitantly with angiotensin II. CONCLUSION: The AT2 receptor affects postnatal cardiac growth possibly via reducing body weight gain and systemic blood pressure. Moreover, it moderately attenuates postnatal vascularization of the heart and modulates the beta adrenergic response of the neonatal heart. These AT2 receptor-mediated effects may be implicated in the physiological maturation process of the heart.