RESUMO
The ability to generate patient-specific human induced pluripotent stem cells (iPSCs) offers a new paradigm for modelling human disease and for individualizing drug testing. Congenital long QT syndrome (LQTS) is a familial arrhythmogenic syndrome characterized by abnormal ion channel function and sudden cardiac death. Here we report the development of a patient/disease-specific human iPSC line from a patient with type-2 LQTS (which is due to the A614V missense mutation in the KCNH2 gene). The generated iPSCs were coaxed to differentiate into the cardiac lineage. Detailed whole-cell patch-clamp and extracellular multielectrode recordings revealed significant prolongation of the action-potential duration in LQTS human iPSC-derived cardiomyocytes (the characteristic LQTS phenotype) when compared to healthy control cells. Voltage-clamp studies confirmed that this action-potential-duration prolongation stems from a significant reduction of the cardiac potassium current I(Kr). Importantly, LQTS-derived cells also showed marked arrhythmogenicity, characterized by early-after depolarizations and triggered arrhythmias. We then used the LQTS human iPSC-derived cardiac-tissue model to evaluate the potency of existing and novel pharmacological agents that may either aggravate (potassium-channel blockers) or ameliorate (calcium-channel blockers, K(ATP)-channel openers and late sodium-channel blockers) the disease phenotype. Our study illustrates the ability of human iPSC technology to model the abnormal functional phenotype of an inherited cardiac disorder and to identify potential new therapeutic agents. As such, it represents a promising paradigm to study disease mechanisms, optimize patient care (personalized medicine), and aid in the development of new therapies.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Células-Tronco Pluripotentes Induzidas/patologia , Síndrome do QT Longo/patologia , Modelos Biológicos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Adulto , Transdiferenciação Celular , Células Cultivadas , Reprogramação Celular/genética , Canal de Potássio ERG1 , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Canais de Potássio Éter-A-Go-Go/química , Canais de Potássio Éter-A-Go-Go/genética , Canais de Potássio Éter-A-Go-Go/metabolismo , Feminino , Fibroblastos/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Síndrome do QT Longo/classificação , Síndrome do QT Longo/tratamento farmacológico , Síndrome do QT Longo/genética , Mutação de Sentido Incorreto/genética , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , Fenótipo , Medicina de Precisão/métodosRESUMO
AIMS: Myocardial cell replacement therapies are hampered by a paucity of sources for human cardiomyocytes and by the expected immune rejection of allogeneic cell grafts. The ability to derive patient-specific human-induced pluripotent stem cells (hiPSCs) may provide a solution to these challenges. We aimed to derive hiPSCs from heart failure (HF) patients, to induce their cardiomyocyte differentiation, to characterize the generated hiPSC-derived cardiomyocytes (hiPSC-CMs), and to evaluate their ability to integrate with pre-existing cardiac tissue. METHODS AND RESULTS: Dermal fibroblasts from two HF patients were reprogrammed by retroviral delivery of Oct4, Sox2, and Klf4 or by using an excisable polycistronic lentiviral vector. The resulting HF-hiPSCs displayed adequate reprogramming properties and could be induced to differentiate into cardiomyocytes with the same efficiency as control hiPSCs (derived from human foreskin fibroblasts). Gene expression and immunostaining studies confirmed the cardiomyocyte phenotype of the differentiating HF-hiPSC-CMs. Multi-electrode array recordings revealed the development of a functional cardiac syncytium and adequate chronotropic responses to adrenergic and cholinergic stimulation. Next, functional integration and synchronized electrical activities were demonstrated between hiPSC-CMs and neonatal rat cardiomyocytes in co-culture studies. Finally, in vivo transplantation studies in the rat heart revealed the ability of the HF-hiPSC-CMs to engraft, survive, and structurally integrate with host cardiomyocytes. CONCLUSIONS: Human-induced pluripotent stem cells can be established from patients with advanced heart failure and coaxed to differentiate into cardiomyocytes, which can integrate with host cardiac tissue. This novel source for patient-specific heart cells may bring a unique value to the emerging field of cardiac regenerative medicine.
Assuntos
Insuficiência Cardíaca/patologia , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Animais , Diferenciação Celular , Sobrevivência Celular , Reprogramação Celular/efeitos dos fármacos , Feminino , Vetores Genéticos , Insuficiência Cardíaca/terapia , Humanos , Células-Tronco Pluripotentes Induzidas/transplante , Cariótipo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/farmacologia , Fator 3 de Transcrição de Octâmero/farmacologia , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição SOXB1/farmacologia , Transgenes , Transplante HeterólogoRESUMO
BACKGROUND: Electroporation is a promising nonthermal ablation method for cardiac arrhythmia treatment. Although initial clinical studies found electroporation pulsed-field ablation (PFA) both safe and efficacious, there are significant knowledge gaps concerning the mechanistic nature and electrophysiological consequences of cardiomyocyte electroporation, contributed by the paucity of suitable human in vitro models. Here, we aimed to establish and characterize a functional in vitro model based on human-induced pluripotent stem cells (hiPSCs)-derived cardiac tissue, and to study the fundamentals of cardiac PFA. METHODS: hiPSC-derived cardiomyocytes were seeded as circular cell sheets and subjected to different PFA protocols. Detailed optical mapping, cellular, and molecular characterizations were performed to study PFA mechanisms and electrophysiological outcomes. RESULTS: PFA generated electrically silenced lesions within the hiPSC-derived cardiac circular cell sheets, resulting in areas of conduction block. Both reversible and irreversible electroporation components were identified. Significant electroporation reversibility was documented within 5 to 15-minutes post-PFA. Irreversibly electroporated regions persisted at 24-hours post-PFA. Per single pulse, high-frequency PFA was less efficacious than standard (monophasic) PFA, whereas increasing pulse-number augmented lesion size and diminished reversible electroporation. PFA augmentation could also be achieved by increasing extracellular Ca2+ levels. Flow-cytometry experiments revealed that regulated cell death played an important role following PFA. Assessing for PFA antiarrhythmic properties, sustainable lines of conduction block could be generated using PFA, which could either terminate or isolate arrhythmic activity in the hiPSC-derived cardiac circular cell sheets. CONCLUSIONS: Cardiac electroporation may be studied using hiPSC-derived cardiac tissue, providing novel insights into PFA temporal and electrophysiological characteristics, facilitating electroporation protocol optimization, screening for potential PFA-sensitizers, and investigating the mechanistic nature of PFA antiarrhythmic properties.
Assuntos
Ablação por Cateter , Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Arritmias Cardíacas/genética , Arritmias Cardíacas/cirurgia , Antiarrítmicos/uso terapêutico , Miócitos Cardíacos/metabolismo , Eletroporação , Ablação por Cateter/métodosRESUMO
Background Optogenetics, using light-sensitive proteins, emerged as a unique experimental paradigm to modulate cardiac excitability. We aimed to develop high-resolution optogenetic approaches to modulate electrical activity in 2- and 3-dimensional cardiac tissue models derived from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. Methods and Results To establish light-controllable cardiac tissue models, opsin-carrying HEK293 cells, expressing the light-sensitive cationic-channel CoChR, were mixed with hiPSC-cardiomyocytes to generate 2-dimensional hiPSC-derived cardiac cell-sheets or 3-dimensional engineered heart tissues. Complex illumination patterns were designed with a high-resolution digital micro-mirror device. Optical mapping and force measurements were used to evaluate the tissues' electromechanical properties. The ability to optogenetically pace and shape the tissue's conduction properties was demonstrated by using single or multiple illumination stimulation sites, complex illumination patterns, or diffuse illumination. This allowed to establish in vitro models for optogenetic-based cardiac resynchronization therapy, where the electrical activation could be synchronized (hiPSC-derived cardiac cell-sheets and engineered heart tissue models) and contractile properties improved (engineered heart tissues). Next, reentrant activity (rotors) was induced in the hiPSC-derived cardiac cell-sheets and engineered heart tissue models through optogenetics programmed- or cross-field stimulations. Diffuse illumination protocols were then used to terminate arrhythmias, demonstrating the potential to study optogenetics cardioversion mechanisms and to identify optimal illumination parameters for arrhythmia termination. Conclusions By combining optogenetics and hiPSC technologies, light-controllable human cardiac tissue models could be established, in which tissue excitability can be modulated in a functional, reversible, and localized manner. This approach may bring a unique value for physiological/pathophysiological studies, for disease modeling, and for developing optogenetic-based cardiac pacing, resynchronization, and defibrillation approaches.
Assuntos
Células-Tronco Pluripotentes Induzidas , Potenciais de Ação/fisiologia , Arritmias Cardíacas , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Optogenética/métodosRESUMO
Heterozygous missense variants of the cardiac ryanodine receptor gene (RYR2) cause catecholaminergic polymorphic ventricular tachycardia (CPVT). These missense variants of RYR2 result in a gain of function of the ryanodine receptors, characterized by increased sensitivity to activation by calcium that results in an increased propensity to develop calcium waves and delayed afterdepolarizations. We have recently detected a nonsense variant in RYR2 in a young patient who suffered an unexplained cardiac arrest. To understand the mechanism by which this variant in RYR2, p.(Arg4790Ter), leads to ventricular arrhythmias, human induced pluripotent stem cells (hiPSCs) harboring the novel nonsense variant in RYR2 were generated and differentiated into cardiomyocytes (RYR2-hiPSC-CMs) and molecular and calcium handling properties were studied. RYR2-hiPSC-CMs displayed significant calcium handling abnormalities at baseline and following treatment with isoproterenol. Treatment with carvedilol and nebivolol resulted in a significant reduction in calcium handling abnormalities in the RYR2-hiPSC-CMs. Expression of the mutant RYR2 allele was confirmed at the mRNA level and partial silencing of the mutant allele resulted in a reduction in calcium handling abnormalities at baseline. The nonsense variant behaves similarly to other gain of function variants in RYR2. Carvedilol and nebivolol may be suitable treatments for patients with gain of function RYR2 variants.
Assuntos
Células-Tronco Pluripotentes Induzidas , Canal de Liberação de Cálcio do Receptor de Rianodina , Cálcio/metabolismo , Sinalização do Cálcio , Carvedilol , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação , Miócitos Cardíacos/metabolismo , Nebivolol/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
Abnormal action potential (AP) properties, as occurs in long or short QT syndromes (LQTS and SQTS, respectively), can cause life-threatening arrhythmias. Optogenetics strategies, utilizing light-sensitive proteins, have emerged as experimental platforms for cardiac pacing, resynchronization, and defibrillation. We tested the hypothesis that similar optogenetic tools can modulate the cardiomyocyte's AP properties, as a potentially novel antiarrhythmic strategy. Healthy control and LQTS/SQTS patient-specific human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were transduced to express the light-sensitive cationic channel channelrhodopsin-2 (ChR2) or the anionic-selective opsin, ACR2. Detailed patch-clamp, confocal-microscopy, and optical mapping studies evaluated the ability of spatiotemporally defined optogenetic protocols to modulate AP properties and prevent arrhythmogenesis in the hiPSC-CMs cell/tissue models. Depending on illumination timing, light-induced ChR2 activation induced robust prolongation or mild shortening of AP duration (APD), while ACR2 activation allowed effective APD shortening. Fine-tuning these approaches allowed for the normalization of pathological AP properties and suppression of arrhythmogenicity in the LQTS/SQTS hiPSC-CM cellular models. We next established a SQTS-hiPSC-CMs-based tissue model of reentrant-arrhythmias using optogenetic cross-field stimulation. An APD-modulating optogenetic protocol was then designed to dynamically prolong APD of the propagating wavefront, completely preventing arrhythmogenesis in this model. This work highlights the potential of optogenetics in studying repolarization abnormalities and in developing novel antiarrhythmic therapies.
Assuntos
Potenciais de Ação/fisiologia , Arritmias Cardíacas/fisiopatologia , Síndrome do QT Longo/fisiopatologia , Miócitos Cardíacos/fisiologia , Channelrhodopsins/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Microscopia Confocal , Miócitos Cardíacos/metabolismo , Opsinas/genética , Imagem Óptica , Optogenética , Técnicas de Patch-ClampRESUMO
Tissue engineering provides unique opportunities for disease modeling, drug testing, and regenerative medicine applications. The use of cell-seeded scaffolds to promote tissue development is the hallmark of the tissue engineering. Among the different types of scaffolds (derived from either natural or synthetic polymers) used in the field, the use of decellularized tissues/organs is specifically attractive. The decellularization process involves the removal of native cells from the original tissue, allowing for the preservation of the three-dimensional (3D) macroscopic and microscopic structures of the tissue and extracellular matrix (ECM) composition. Following recellularization, the resulting scaffold provides the seeded cells with the appropriate biological signals and mechanical properties of the original tissue. Here, we describe different methods to create viable scaffolds from decellularized heart and liver as useful tools to study and exploit ECM biological key factors for the generation of engineered tissues with enhanced regenerative properties.
Assuntos
Derme Acelular/metabolismo , Medicina Regenerativa/métodos , Engenharia Tecidual/métodos , Animais , Matriz Extracelular/química , Coração/crescimento & desenvolvimento , Hepatócitos/citologia , Fígado/crescimento & desenvolvimento , Miócitos Cardíacos/citologia , CoelhosRESUMO
BACKGROUND: The ability to derive human induced pluripotent stem (hiPS) cell lines by reprogramming of adult fibroblasts with a set of transcription factors offers unique opportunities for basic and translational cardiovascular research. In the present study, we aimed to characterize the cardiomyocyte differentiation potential of hiPS cells and to study the molecular, structural, and functional properties of the generated hiPS-derived cardiomyocytes. METHODS AND RESULTS: Cardiomyocyte differentiation of the hiPS cells was induced with the embryoid body differentiation system. Gene expression studies demonstrated that the cardiomyocyte differentiation process of the hiPS cells was characterized by an initial increase in mesoderm and cardiomesoderm markers, followed by expression of cardiac-specific transcription factors and finally by cardiac-specific structural genes. Cells in the contracting embryoid bodies were stained positively for cardiac troponin-I, sarcomeric alpha-actinin, and connexin-43. Reverse-transcription polymerase chain reaction studies demonstrated the expression of cardiac-specific sarcomeric proteins and ion channels. Multielectrode array recordings established the development of a functional syncytium with stable pacemaker activity and action potential propagation. Positive and negative chronotropic responses were induced by application of isoproterenol and carbamylcholine, respectively. Administration of quinidine, E4031 (I(Kr) blocker), and chromanol 293B (I(Ks) blocker) significantly affected repolarization, as manifested by prolongation of the local field potential duration. CONCLUSIONS: hiPS cells can differentiate into myocytes with cardiac-specific molecular, structural, and functional properties. These results, coupled with the potential of this technology to generate patient-specific hiPS lines, hold great promise for the development of in vitro models of cardiac genetic disorders, for drug discovery and testing, and for the emerging field of cardiovascular regenerative medicine.
Assuntos
Diferenciação Celular/fisiologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Adulto , Animais , Linhagem Celular , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/fisiologia , Humanos , CamundongosRESUMO
The objective of the current study was to characterize calcium handling in developing human embryonic stem cell-derived cardiomyocytes (hESC-CMs). To this end, real-time polymerase chain reaction (PCR), immunocytochemistry, whole-cell voltage-clamp, and simultaneous patch-clamp/laser scanning confocal calcium imaging and surface membrane labeling with di-8-aminonaphthylethenylpridinium were used. Immunostaining studies in the hESC-CMs demonstrated the presence of the sarcoplasmic reticulum (SR) calcium release channels, ryanodine receptor-2, and inositol-1,4,5-trisphosphate (IP3) receptors. Store calcium function was manifested as action-potential-induced calcium transients. Time-to-target plots showed that these action-potential-initiated calcium transients traverse the width of the cell via a propagated wave of intracellular store calcium release. The hESC-CMs also exhibited local calcium events ("sparks") that were localized to the surface membrane. The presence of caffeine-sensitive intracellular calcium stores was manifested following application of focal, temporally limited puffs of caffeine in three different age groups: early-stage (with the initiation of beating), intermediate-stage (10 days post-beating [dpb]), and late-stage (30-40 dpb) hESC-CMs. Calcium store load gradually increased during in vitro maturation. Similarly, ryanodine application decreased the amplitude of the spontaneous calcium transients. Interestingly, the expression and function of an IP3-releasable calcium pool was also demonstrated in the hESC-CMs in experiments using caged-IP3 photolysis and antagonist application (2 microM 2-Aminoethoxydiphenyl borate). In summary, our study establishes the presence of a functional SR calcium store in early-stage hESC-CMs and shows a unique pattern of calcium handling in these cells. This study also stresses the importance of the functional characterization of hESC-CMs both for developmental studies and for the development of future myocardial cell replacement strategies. Disclosure of potential conflicts of interest is found at the end of this article.
Assuntos
Cálcio/metabolismo , Células-Tronco Embrionárias/citologia , Miócitos Cardíacos/citologia , Potenciais de Ação , Cafeína/farmacologia , Membrana Celular/metabolismo , Humanos , Imuno-Histoquímica/métodos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Microscopia Confocal , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , Inibidores de Fosfodiesterase/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMO
Transplantation of a tissue-engineered heart muscle represents a novel experimental therapeutic paradigm for myocardial diseases. However, this strategy has been hampered by the lack of sources for human cardiomyocytes and by the scarce vasculature in the ischemic area limiting the engraftment and survival of the transplanted muscle. Beyond the necessity of endothelial capillaries for the delivery of oxygen and nutrients to the grafted muscle tissue, interactions between endothelial and cardiomyocyte cells may also play a key role in promoting cell survival and proliferation. In the present study, we describe the formation of synchronously contracting engineered human cardiac tissue derived from human embryonic stem cells containing endothelial vessel networks. The 3D muscle consisted of cardiomyocytes, endothelial cells (ECs), and embryonic fibroblasts (EmFs). The formed vessels were further stabilized by the presence of mural cells originating from the EmFs. The presence of EmFs decreased EC death and increased EC proliferation. Moreover, the presence of endothelial capillaries augmented cardiomyocyte proliferation and did not hamper cardiomyocyte orientation and alignment. Immunostaining, ultrastructural analysis (using transmission electron microscopy), RT-PCR, pharmacological, and confocal laser calcium imaging studies demonstrated the presence of cardiac-specific molecular, ultrastructural, and functional properties of the generated tissue constructs with synchronous activity mediated by action potential propagation through gap junctions. In summary, this is the first report of the construction of 3D vascularized human cardiac tissue that may have unique applications for studies of cardiac development, function, and tissue replacement therapy.
Assuntos
Vasos Coronários/citologia , Células-Tronco Embrionárias/citologia , Miocárdio/citologia , Engenharia Tecidual/métodos , Técnicas de Cultura de Células , Proliferação de Células , Células Cultivadas , Vasos Coronários/ultraestrutura , Células-Tronco Embrionárias/ultraestrutura , Humanos , Microscopia Confocal , Miocárdio/ultraestrutura , Miócitos Cardíacos/citologia , Miócitos Cardíacos/ultraestrutura , Neovascularização Fisiológica/fisiologiaRESUMO
BACKGROUND: The short QT syndrome (SQTS) is an inherited arrhythmogenic syndrome characterized by abnormal ion channel function, life-threatening arrhythmias, and sudden cardiac death. OBJECTIVES: The purpose of this study was to establish a patient-specific human-induced pluripotent stem cell (hiPSC) model of the SQTS, and to provide mechanistic insights into its pathophysiology and therapy. METHODS: Patient-specific hiPSCs were generated from a symptomatic SQTS patient carrying the N588K mutation in the KCNH2 gene, differentiated into cardiomyocytes, and compared with healthy and isogenic (established by CRISPR/Cas9-based mutation correction) control hiPSC-derived cardiomyocytes (hiPSC-CMs). Patch-clamp was used to evaluate action-potential (AP) and IKr current properties at the cellular level. Conduction and arrhythmogenesis were studied at the tissue level using confluent 2-dimensional hiPSC-derived cardiac cell sheets (hiPSC-CCSs) and optical mapping. RESULTS: Intracellular recordings demonstrated shortened action-potential duration (APD) and abbreviated refractory period in the SQTS-hiPSC-CMs. Similarly, voltage- and AP-clamp recordings revealed increased IKr current density due to attenuated inactivation, primarily in the AP plateau phase. Optical mapping of the SQTS-hiPSC-CCSs revealed shortened APD, impaired APD-rate adaptation, abbreviated wavelength of excitation, and increased inducibility of sustained spiral waves. Phase-mapping analysis revealed accelerated and stabilized rotors manifested by increased rotor rotation frequency, increased rotor curvature, decreased core meandering, and increased rotor complexity. Application of quinidine and disopyramide, but not sotalol, normalized APD and suppressed arrhythmia induction. CONCLUSIONS: A novel hiPSC-based model of the SQTS was established at both the cellular and tissue levels. This model recapitulated the disease phenotype in the culture dish and provided important mechanistic insights into arrhythmia mechanisms in the SQTS and its treatment.
Assuntos
Arritmias Cardíacas , Miócitos Cardíacos/metabolismo , Potenciais de Ação , Antiarrítmicos/farmacologia , Arritmias Cardíacas/diagnóstico , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/prevenção & controle , Células Cultivadas , Canal de Potássio ERG1/genética , Humanos , Células-Tronco Pluripotentes Induzidas , Mutação , Técnicas de Patch-Clamp , Modelagem Computacional Específica para o PacienteRESUMO
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).
Assuntos
Arritmias Cardíacas/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos , Matriz Extracelular/metabolismo , Coração/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Modelos Cardiovasculares , Miócitos Cardíacos/citologia , Engenharia Tecidual/métodos , Potenciais de Ação/efeitos dos fármacos , Animais , Arritmias Cardíacas/patologia , Cálcio/metabolismo , Fármacos Cardiovasculares/farmacologia , Modelos Animais de Doenças , Matriz Extracelular/efeitos dos fármacos , Humanos , Hidrogéis/farmacologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Especificidade de Órgãos , SuínosRESUMO
Human embryonic stem cells (hESC) are pluripotent lines that can differentiate in vitro into cell derivatives of all three germ layers, including cardiomyocytes. Successful application of these unique cells in the areas of cardiovascular research and regenerative medicine has been hampered by difficulties in identifying and selecting specific cardiac progenitor cells from the mixed population of differentiating cells. We report the generation of stable transgenic hESC lines, using lentiviral vectors, and single-cell clones that express a reporter gene (eGFP) under the transcriptional control of a cardiac-specific promoter (the human myosin light chain-2V promoter). Our results demonstrate the appearance of eGFP-expressing cells during the differentiation of the hESC as embryoid bodies (EBs) that can be identified and sorted using FACS (purity>95%, viability>85%). The eGFP-expressing cells were stained positively for cardiac-specific proteins (>93%), expressed cardiac-specific genes, displayed cardiac-specific action-potentials, and could form stable myocardial cell grafts following in vivo cell transplantation. The generation of these transgenic hESC lines may be used to identify and study early cardiac precursors for developmental studies, to robustly quantify the extent of cardiomyocyte differentiation, to label the cells for in vivo grafting, and to allow derivation of purified cell populations of cardiomyocytes for future myocardial cell therapy strategies.
Assuntos
Células-Tronco Embrionárias/citologia , Coração/fisiologia , Células Musculares/fisiologia , Diferenciação Celular , Linhagem Celular , Células Clonais , Primers do DNA , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Coração/embriologia , Humanos , Células Musculares/citologia , Miocárdio/citologia , TransfecçãoRESUMO
Fulfilling the potential of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes for studying conduction and arrhythmogenesis requires development of multicellular models and methods for long-term repeated tissue phenotyping. We generated confluent hiPSC-derived cardiac cell sheets (hiPSC-CCSs), expressing the genetically encoded voltage indicator ArcLight. ArcLight-based optical mapping allowed generation of activation and action-potential duration (APD) maps, which were validated by mapping the same hiPSC-CCSs with the voltage-sensitive dye, Di-4-ANBDQBS. ArcLight mapping allowed long-term assessment of electrical remodeling in the hiPSC-CCSs and evaluation of drug-induced conduction slowing (carbenoxolone, lidocaine, and quinidine) and APD prolongation (quinidine and dofetilide). The latter studies also enabled step-by-step depiction of drug-induced arrhythmogenesis ("torsades de pointes in the culture dish") and its prevention by MgSO4 and rapid pacing. Phase-mapping analysis allowed biophysical characterization of spiral waves induced in the hiPSC-CCSs and their termination by electrical cardioversion and overdrive pacing. In conclusion, ArcLight mapping of hiPSC-CCSs provides a powerful tool for drug testing and arrhythmia investigation.
Assuntos
Biomarcadores , Expressão Gênica , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Potenciais de Ação , Arritmias Cardíacas/tratamento farmacológico , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Humanos , Modelos Biológicos , Imagem Molecular , Miócitos Cardíacos/efeitos dos fármacos , Fenetilaminas , SulfonamidasRESUMO
Cell therapy is emerging as a promising strategy for myocardial repair. This approach is hampered, however, by the lack of sources for human cardiac tissue and by the absence of direct evidence for functional integration of donor cells into host tissues. Here we investigate whether cells derived from human embryonic stem (hES) cells can restore myocardial electromechanical properties. Cardiomyocyte cell grafts were generated from hES cells in vitro using the embryoid body differentiating system. This tissue formed structural and electromechanical connections with cultured rat cardiomyocytes. In vivo integration was shown in a large-animal model of slow heart rate. The transplanted hES cell-derived cardiomyocytes paced the hearts of swine with complete atrioventricular block, as assessed by detailed three-dimensional electrophysiological mapping and histopathological examination. These results demonstrate the potential of hES-cell cardiomyocytes to act as a rate-responsive biological pacemaker and for future myocardial regeneration strategies.
Assuntos
Estimulação Cardíaca Artificial/métodos , Bloqueio Cardíaco/fisiopatologia , Bloqueio Cardíaco/cirurgia , Sistema de Condução Cardíaco/fisiopatologia , Contração Miocárdica , Miócitos Cardíacos , Transplante de Células-Tronco/métodos , Animais , Animais Recém-Nascidos , Mapeamento Potencial de Superfície Corporal , Diferenciação Celular , Sobrevivência de Enxerto , Bloqueio Cardíaco/diagnóstico , Humanos , Ratos , Ratos Sprague-Dawley , Suínos , Resultado do TratamentoRESUMO
BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia type 2 (CPVT2) results from autosomal recessive CASQ2 mutations, causing abnormal Ca2+-handling and malignant ventricular arrhythmias. We aimed to establish a patient-specific human induced pluripotent stem cell (hiPSC) model of CPVT2 and to use the generated hiPSC-derived cardiomyocytes to gain insights into patient-specific disease mechanism and pharmacotherapy. METHODS AND RESULTS: hiPSC cardiomyocytes were derived from a CPVT2 patient (D307H-CASQ2 mutation) and from healthy controls. Laser-confocal Ca2+ and voltage imaging showed significant Ca2+-transient irregularities, marked arrhythmogenicity manifested by early afterdepolarizations and triggered arrhythmias, and reduced threshold for store overload-induced Ca2+-release events in the CPVT2-hiPSC cardiomyocytes when compared with healthy control cells. Pharmacological studies revealed the prevention of adrenergic-induced arrhythmias by ß-blockers (propranolol and carvedilol), flecainide, and the neuronal sodium-channel blocker riluzole; a direct antiarrhythmic action of carvedilol (independent of its α/ß-adrenergic blocking activity), flecainide, and riluzole; and suppression of abnormal Ca2+ cycling by the ryanodine stabilizer JTV-519 and carvedilol. Mechanistic insights were gained on the different antiarrhythmic actions of the aforementioned drugs, with carvedilol and JTV-519 (but not flecainide or riluzole) acting primarily through sarcoplasmic reticulum stabilization. Finally, comparable outcomes were found between flecainide and labetalol antiarrhythmic effects in vitro and the clinical results in the same patient. CONCLUSIONS: These results demonstrate the ability of hiPSCs cardiomyocytes to recapitulate CPVT2 disease phenotype and drug response in the culture dish, to provide novel insights into disease and drug therapy mechanisms, and potentially to tailor patient-specific drug therapy.
Assuntos
Antiarrítmicos/farmacologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Medicina de Precisão , Taquicardia Ventricular/tratamento farmacológico , Potenciais de Ação , Agonistas Adrenérgicos/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Calsequestrina/genética , Calsequestrina/metabolismo , Estudos de Casos e Controles , Linhagem Celular , Relação Dose-Resposta a Droga , Predisposição Genética para Doença , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Mutação , Miócitos Cardíacos/metabolismo , Seleção de Pacientes , Fenótipo , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , Taquicardia Ventricular/genética , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/fisiopatologia , Fatores de Tempo , Adulto JovemRESUMO
The advent of the human-induced pluripotent stem cell (hiPSC) technology has transformed biomedical research, providing new tools for human disease modeling, drug development, and regenerative medicine. To fulfill its unique potential in the cardiovascular field, efficient methods should be developed for high-resolution, large-scale, long-term, and serial functional cellular phenotyping of hiPSC-derived cardiomyocytes (hiPSC-CMs). To achieve this goal, we combined the hiPSC technology with genetically encoded voltage (ArcLight) and calcium (GCaMP5G) fluorescent indicators. Expression of ArcLight and GCaMP5G in hiPSC-CMs permitted to reliably follow changes in transmembrane potential and intracellular calcium levels, respectively. This allowed monitoring short- and long-term changes in action-potential and calcium-handling properties and the development of arrhythmias in response to several pharmaceutical agents and in hiPSC-CMs derived from patients with different inherited arrhythmogenic syndromes. Combining genetically encoded fluorescent reporters with hiPSC-CMs may bring a unique value to the study of inherited disorders, developmental biology, and drug development and testing.
Assuntos
Potenciais de Ação , Cálcio/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Arritmias Cardíacas/metabolismo , Cálcio/análise , Diferenciação Celular , Células Cultivadas , Expressão Gênica , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Miócitos Cardíacos/metabolismo , Imagem Óptica/métodos , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Transdução Genética , TransgenesRESUMO
BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a primary heart muscle disorder resulting from desmosomal protein mutations. ARVC is characterized pathologically by fibrofatty infiltration and clinically by arrhythmias and sudden cardiac death. We aimed to establish a patient-/disease-specific human induced pluripotent stem cell (hiPSC) model of ARVC. METHODS AND RESULTS: Dermal fibroblasts were obtained from 2 patients with ARVC with plakophilin-2 (PKP2) mutations, reprogrammed to generate hiPSCs, coaxed to differentiate into cardiomyocytes (CMs), and then compared with healthy control hiPSC-derived CMs (hiPSC-CMs). Real-time polymerase chain reaction showed a significant decrease in the expression of PKP2 in the ARVC-hiPSC-CMs. Immunostainings revealed reduced densities of PKP2, the associated desmosomal protein plakoglobin, and the gap-junction protein connexin-43. Electrophysiological assessment demonstrated prolonged field potential rise time in the ARVC-hiPSC-CMs. Transmission electron microscopy identified widened and distorted desmosomes in the ARVC-hiPSC-CMs. Clusters of lipid droplets were identified in the ARVC-CMs that displayed the more severe desmosomal pathology. This finding was associated with upregulation of the proadipogenic transcription factor peroxisome proliferator-activated receptor-γ. Exposure of the cells to apidogenic stimuli augmented desmosomal distortion and lipid accumulation. The latter phenomenon was prevented by application of a specific inhibitor of glycogen synthase kinase 3ß (6-bromoindirubin-3'-oxime). CONCLUSIONS: This study highlights the unique potential of the hiPSC technology for modeling inherited cardiac disorders in general and ARVC specifically. The hiPSC-CMs were demonstrated to recapitulate the ARVC phenotype in the dish, provide mechanistic insights into early disease pathogenesis, and provide a unique platform for drug discovery and testing in this disorder.
Assuntos
Displasia Arritmogênica Ventricular Direita/metabolismo , Fibroblastos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Cardiovasculares , Apoptose , Displasia Arritmogênica Ventricular Direita/genética , Displasia Arritmogênica Ventricular Direita/fisiopatologia , Diferenciação Celular , Células Cultivadas , Conexina 43/metabolismo , Derme/metabolismo , Derme/patologia , Desmossomos/efeitos dos fármacos , Desmossomos/metabolismo , Eletrocardiografia , Fibroblastos/fisiologia , Expressão Gênica , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Humanos , Imuno-Histoquímica , Indóis/farmacologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Microscopia Eletrônica de Transmissão , Mutação , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Miócitos Cardíacos/ultraestrutura , Oximas/farmacologia , Placofilinas/genética , Placofilinas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , gama Catenina/metabolismoRESUMO
OBJECTIVES: The goal of this study was to establish a patient-specific human-induced pluripotent stem cells (hiPSCs) model of catecholaminergic polymorphic ventricular tachycardia (CPVT). BACKGROUND: CPVT is a familial arrhythmogenic syndrome characterized by abnormal calcium (Ca(2+)) handling, ventricular arrhythmias, and sudden cardiac death. METHODS: Dermal fibroblasts were obtained from a CPVT patient due to the M4109R heterozygous point RYR2 mutation and reprogrammed to generate the CPVT-hiPSCs. The patient-specific hiPSCs were coaxed to differentiate into the cardiac lineage and compared with healthy control hiPSCs-derived cardiomyocytes (hiPSCs-CMs). RESULTS: Intracellular electrophysiological recordings demonstrated the development of delayed afterdepolarizations in 69% of the CPVT-hiPSCs-CMs compared with 11% in healthy control cardiomyocytes. Adrenergic stimulation by isoproterenol (1 µM) or forskolin (5 µM) increased the frequency and magnitude of afterdepolarizations and also led to development of triggered activity in the CPVT-hiPSCs-CMs. In contrast, flecainide (10 µM) and thapsigargin (10 µM) eliminated all afterdepolarizations in these cells. The latter finding suggests an important role for internal Ca(2+) stores in the pathogenesis of delayed afterdepolarizations. Laser-confocal Ca(2+) imaging revealed significant whole-cell [Ca(2+)] transient irregularities (frequent local and large-storage Ca(2+)-release events, broad and double-humped transients, and triggered activity) in the CPVT cardiomyocytes that worsened with adrenergic stimulation and Ca(2+) overload and improved with beta-blockers. Store-overload-induced Ca(2+) release was also identified in the hiPSCs-CMs and the threshold for such events was significantly reduced in the CPVT cells. CONCLUSIONS: This study highlights the potential of hiPSCs for studying inherited arrhythmogenic syndromes, in general, and CPVT specifically. As such, it represents a promising paradigm to study disease mechanisms, optimize patient care, and aid in the development of new therapies.