RESUMEN

BACKGROUND: Despite a rigorous screening process, including cardiac catheterization, a subset of patients with a single right ventricle (SRV) demonstrates suboptimal short-term outcomes after the Fontan operation. The goal of this study was to perform a comprehensive assessment of diastolic function in pre-Fontan patients with an SRV using invasive reference-standard measures and determine their associations with post-Fontan outcomes. METHODS AND RESULTS: Children aged 2 to 6 years with SRV physiology undergoing pre-Fontan heart catheterization were recruited prospectively. Patients were divided into those who had an optimal or suboptimal outcome. A suboptimal outcome was defined as length of stay ≥14 days or heart transplant/cardiac death in first year after Fontan. Patients underwent pressure-volume loop analysis using reference-standard methods. The measure of ventricular stiffness, ß, was obtained via preload reduction. Cardiac magnetic resonance imaging for extracellular volume and serum draws for matrix metalloproteinase activity were performed. Of 19 patients with an SRV, 9 (47%) had a suboptimal outcome. Mean age was 4.2±0.7 years. Patients with suboptimal outcomes had lower ventricular stiffness (0.021 [0.009-0.049] versus 0.090 [0.031-0.118] mL-1; P=0.02), lower extracellular volume (25% [28%-32%] versus 31% [28%-33%]; P=0.02), and lower matrix metalloproteinase-2 (90 [79-104] versus 108 [79-128] ng/mL; P=0.01) compared with patients with optimal outcomes. The only invasive measure that had an association with suboptimal outcome was ß (P=0.038). CONCLUSIONS: Patients with an SRV with suboptimal outcome after the Fontan operation had lower ventricular stiffness and evidence of maladaptive extracellular matrix metabolism compared with patients with optimal outcome. This appears to be a novel phenotype that may have important clinical implications and requires further study.

Asunto(s)

Procedimiento de Fontan , Ventrículos Cardíacos , Fenotipo , Humanos , Procedimiento de Fontan/efectos adversos , Preescolar , Masculino , Femenino , Niño , Ventrículos Cardíacos/fisiopatología , Ventrículos Cardíacos/diagnóstico por imagen , Ventrículos Cardíacos/anomalías , Estudios Prospectivos , Resultado del Tratamiento , Cateterismo Cardíaco , Función Ventricular Derecha/fisiología , Trasplante de Corazón , Metaloproteinasa 2 de la Matriz/sangre , Corazón Univentricular/cirugía , Corazón Univentricular/fisiopatología , Corazón Univentricular/diagnóstico por imagen , Cardiopatías Congénitas/cirugía , Cardiopatías Congénitas/fisiopatología , Factores de Tiempo

RESUMEN

The spleen is an important mediator of both adaptive and innate immunity. As such, attempts to modulate the immune response provided by the spleen may be conducive to improved outcomes for numerous diseases throughout the body. Here, biomimicry is used to rationally design nanomaterials capable of splenic retention and immunomodulation for the treatment of disease in a distant organ, the postinfarct heart. Engineered senescent erythrocyte-derived nanotheranostic (eSENTs) are generated, demonstrating significant uptake by the immune cells of the spleen including T and B cells, as well as monocytes and macrophages. When loaded with suberoylanilide hydroxamic acid (SAHA), the nanoagents exhibit a potent therapeutic effect, reducing infarct size by 14% at 72 h postmyocardial infarction when given as a single intravenous dose 2 h after injury. These results are supportive of the hypothesis that RBC-derived biomimicry may provide new approaches for the targeted modulation of the pathological processes involved in myocardial infarction, thus further experiments to decisively confirm the mechanisms of action are currently underway. This novel concept may have far-reaching applicability for the treatment of a number of both acute and chronic conditions where the immune responses are either stimulated or suppressed by the splenic (auto)immune milieu.

Asunto(s)

Biomimética , Infarto del Miocardio , Humanos , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/patología , Corazón , Inmunidad Innata , Inmunomodulación

RESUMEN

Human cardiac organoids hold remarkable potential for cardiovascular disease modeling and human pluripotent stem cell-derived cardiomyocyte (hPSC-CM) transplantation. Here, we show cardiac organoids engineered with electrically conductive silicon nanowires (e-SiNWs) significantly enhance the therapeutic efficacy of hPSC-CMs to treat infarcted hearts. We first demonstrated the biocompatibility of e-SiNWs and their capacity to improve cardiac microtissue engraftment in healthy rat myocardium. Nanowired human cardiac organoids were then engineered with hPSC-CMs, nonmyocyte supporting cells, and e-SiNWs. Nonmyocyte supporting cells promoted greater ischemia tolerance of cardiac organoids, and e-SiNWs significantly improved electrical pacing capacity. After transplantation into ischemia/reperfusion-injured rat hearts, nanowired cardiac organoids significantly improved contractile development of engrafted hPSC-CMs, induced potent cardiac functional recovery, and reduced maladaptive left ventricular remodeling. Compared to contemporary studies with an identical injury model, greater functional recovery was achieved with a 20-fold lower dose of hPSC-CMs, revealing therapeutic synergy between conductive nanomaterials and human cardiac organoids for efficient heart repair.

Asunto(s)

Células Madre Pluripotentes Inducidas , Infarto del Miocardio , Humanos , Ratas , Animales , Diferenciación Celular , Miocardio , Isquemia , Infarto del Miocardio/terapia , Organoides

RESUMEN

Acute cardiac injuries occur in 20%-25% of hospitalized COVID-19 patients. Herein, we demonstrate that human cardiac organoids (hCOs) are a viable platform to model the cardiac injuries caused by COVID-19 hyperinflammation. As IL-1ß is an upstream cytokine and a core COVID-19 signature cytokine, it was used to stimulate hCOs to induce the release of a milieu of proinflammatory cytokines that mirror the profile of COVID-19 cytokine storm. The IL-1ß treated hCOs recapitulated transcriptomic, structural, and functional signatures of COVID-19 hearts. The comparison of IL-1ß treated hCOs with cardiac tissue from COVID-19 autopsies illustrated the critical roles of hyper-inflammation in COVID-19 cardiac insults and indicated the cardioprotective effects of endothelium. The IL-1ß treated hCOs thus provide a defined and robust model to assess the efficacy and potential side effects of immunomodulatory drugs, as well as the reversibility of COVID-19 cardiac injuries at baseline and simulated exercise conditions.

Asunto(s)

COVID-19 , Síndrome de Liberación de Citoquinas , Cardiopatías , COVID-19/complicaciones , Síndrome de Liberación de Citoquinas/virología , Citocinas/metabolismo , Cardiopatías/virología , Humanos , Modelos Biológicos , Organoides

RESUMEN

Acute cardiac injuries occur in 20-25% of hospitalized COVID-19 patients. Despite urgent needs, there is a lack of 3D organotypic models of COVID-19 hearts for mechanistic studies and drug testing. Herein, we demonstrate that human cardiac organoids (hCOs) are a viable platform to model the cardiac injuries caused by COVID-19 hyperinflammation. As IL-1ßis an upstream cytokine and a core COVID-19 signature cytokine, it was used to stimulate hCOs to induce the release of a milieu of proinflammatory cytokines that mirror the profile of COVID-19 cytokine storm. The IL-1 ß treated hCOs recapitulated transcriptomic, structural, and functional signatures of COVID-19 hearts. The comparison of IL-1ß treated hCOs with cardiac tissue from COVID-19 autopsies illustrated the critical roles of hyper-inflammation in COVID-19 cardiac insults and indicated the cardioprotective effects of endothelium. The IL-1ß treated hCOs also provide a viable model to assess the efficacy and potential side effects of immunomodulatory drugs, as well as the reversibility of COVID-19 cardiac injuries at baseline and simulated exercise conditions.

RESUMEN

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been widely used for disease modeling and drug cardiotoxicity screening. To this end, we recently developed human cardiac organoids (hCOs) for modeling human myocardium. Here, we perform a transcriptomic analysis of various in vitro hiPSC-CM platforms (2D iPSC-CM, 3D iPSC-CM and hCOs) to deduce the strengths and limitations of these in vitro models. We further compared iPSC-CM models to human myocardium samples. Our data show that the 3D in vitro environment of 3D hiPSC-CMs and hCOs stimulates the expression of genes associated with tissue formation. The hCOs demonstrated diverse physiologically relevant cellular functions compared to the hiPSC-CM only models. Including other cardiac cell types within hCOs led to more transcriptomic similarities to adult myocardium. hCOs lack matured cardiomyocytes and immune cells, which limits a complete replication of human adult myocardium. In conclusion, 3D hCOs are transcriptomically similar to myocardium, and future developments of engineered 3D cardiac models would benefit from diversifying cell populations, especially immune cells.

Asunto(s)

Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Organoides/metabolismo , Transcriptoma , Adulto , Células Cultivadas , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Miocardio/citología , Miocitos Cardíacos/citología , Organoides/citología

RESUMEN

Prevascularized 3D microtissues have been shown to be an effective cell delivery vehicle for cardiac repair. To this end, our lab has explored the development of self-organizing, prevascularized human cardiac organoids by co-seeding human cardiomyocytes with cardiac fibroblasts, endothelial cells, and stromal cells into agarose microwells. We hypothesized that this prevascularization process is facilitated by the endogenous upregulation of hypoxia-inducible factor (HIF) pathway in the avascular 3D microtissues. In this study, we used Molidustat, a selective PHD (prolyl hydroxylase domain enzymes) inhibitor that stabilizes HIF-α, to treat human cardiac organoids, which resulted in 150 ± 61% improvement in endothelial expression (CD31) and 220 ± 20% improvement in the number of lumens per organoids. We hypothesized that the improved endothelial expression seen in Molidustat treated human cardiac organoids was dependent upon upregulation of VEGF, a well-known downstream target of HIF pathway. Through the use of immunofluorescent staining and ELISA assays, we determined that Molidustat treatment improved VEGF expression of non-endothelial cells and resulted in improved co-localization of supporting cell types and endothelial structures. We further demonstrated that Molidustat treated human cardiac organoids maintain cardiac functionality. Lastly, we showed that Molidustat treatment improves survival of cardiac organoids when exposed to both hypoxic and ischemic conditions in vitro. For the first time, we demonstrate that targeted HIF-α stabilization provides a robust strategy to improve endothelial expression and lumen formation in cardiac microtissues, which will provide a powerful framework for prevascularization of various microtissues in developing successful cell transplantation therapies.

Asunto(s)

Fibroblastos/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Miocitos Cardíacos/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Organoides , Pirazoles/farmacología , Triazoles/farmacología , Técnicas de Cocultivo , Humanos , Organoides/irrigación sanguínea , Organoides/metabolismo

RESUMEN

Heart failure (HF) has traditionally been defined by symptoms of fluid accumulation and poor perfusion, but it is now recognized that specific HF classifications hold prognostic and therapeutic relevance. Specifically, HF with reduced ejection fraction is characterized by reduced left ventricular systolic pump function and dilation and HF with preserved ejection fraction is characterized primarily by abnormal left ventricular filling (diastolic failure) with relatively preserved left ventricular systolic function. These forms of HF are distributed equally among patients with HF and likely require distinctly different strategies to mitigate the morbidity, mortality, and medical resource utilization of this disease. In particular, HF is a significant medical issue within the US Department of Veterans Affairs (VA) hospital system and constitutes a major translational research priority for the VA. Because a common underpinning of both HF with reduced ejection fraction and HF with preserved ejection fraction seems to be changes in the structure and function of the myocardial extracellular matrix, a conference was convened sponsored by the VA, entitled, "Targeting Myocardial Fibrosis in Heart Failure" to explore the extracellular matrix as a potential therapeutic target and to propose specific research directions. The conference was conceptually framed around the hypothesis that although HF with reduced ejection fraction and HF with preserved ejection fraction clearly have distinct mechanisms, they may share modifiable pathways and biological mediators in common. Inflammation and extracellular matrix were identified as major converging themes. A summary of our discussion on unmet challenges and possible solutions to move the field forward, as well as recommendations for future research opportunities, are provided.

Asunto(s)

Insuficiencia Cardíaca , Disfunción Ventricular Izquierda , Diástole , Fibrosis , Insuficiencia Cardíaca/epidemiología , Insuficiencia Cardíaca/terapia , Humanos , Volumen Sistólico , Función Ventricular Izquierda

RESUMEN

Environmental factors are the largest contributors to cardiovascular disease. Here we show that cardiac organoids that incorporate an oxygen-diffusion gradient and that are stimulated with the neurotransmitter noradrenaline model the structure of the human heart after myocardial infarction (by mimicking the infarcted, border and remote zones), and recapitulate hallmarks of myocardial infarction (in particular, pathological metabolic shifts, fibrosis and calcium handling) at the transcriptomic, structural and functional levels. We also show that the organoids can model hypoxia-enhanced doxorubicin cardiotoxicity. Human organoids that model diseases with non-genetic pathological factors could help with drug screening and development.

Asunto(s)

Evaluación Preclínica de Medicamentos/métodos , Corazón/efectos de los fármacos , Modelos Cardiovasculares , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Organoides/efectos de los fármacos , Cardiotoxicidad/metabolismo , Cardiotoxicidad/patología , Desarrollo de Medicamentos , Humanos , Infarto del Miocardio/inducido químicamente , Infarto del Miocardio/genética , Organoides/metabolismo , Organoides/patología , Oxígeno/metabolismo

RESUMEN

Ischemia reperfusion injury (I/R injury) contributes significantly to morbidity and mortality following myocardial infarction (MI). Although rapid reperfusion of the ischemic myocardium was established decades ago as a highly beneficial therapy for MI, significant cell death still occurs after the onset of reperfusion. Mitochondrial dysfunction is closely associated with I/R injury, resulting in the uncontrolled production of reactive oxygen species (ROS). Considerable efforts have gone into understanding the metabolic perturbations elicited by I/R injury. Recent work has identified the critical role of reversible protein acetylation in maintaining normal mitochondrial biologic function and energy metabolism both in the normal heart and during I/R injury. Several studies have shown that modification of class I HDAC and/or Sirtuin (Sirt) activity is cardioprotective in the setting of I/R injury. A better understanding of the role of these metabolic pathways in reperfusion injury and their regulation by reversible protein acetylation presents a promising way forward in improving the treatment of cardiac reperfusion injury. Here we briefly review some of what is known about how acetylation regulates mitochondrial metabolism and how it relates to I/R injury.

Asunto(s)

Metabolismo Energético , Infarto del Miocardio/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión/metabolismo , Acetilación , Animales , Humanos , Ratones , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/patología , Infarto del Miocardio/patología , Daño por Reperfusión Miocárdica/patología , Miocardio/metabolismo , Miocardio/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Especies Reactivas de Oxígeno/metabolismo , Daño por Reperfusión/patología

RESUMEN

AIMS: Following an acute myocardial infarction (MI) the extracellular matrix (ECM) undergoes remodeling in order to prevent dilation of the infarct area and maintain cardiac output. Excessive and prolonged inflammation following an MI exacerbates adverse ventricular remodeling. Macrophages are an integral part of the inflammatory response that contribute to this remodeling. Treatment with histone deacetylase (HDAC) inhibitors preserves LV function and myocardial remodeling in the post-MI heart. This study tested whether inhibition of HDAC activity resulted in preserving post-MI LV function through the regulation of macrophage phenotype and early resolution of inflammation. METHODS AND RESULTS: HDAC inhibition does not affect the recruitment of CD45+ leukocytes, CD45+/CD11b+ inflammatory monocytes or CD45+/CD11b+CD86+ inflammatory macrophages for the first 3 days following infarct. Further, HDAC inhibition does not change the high expression level of the inflammatory cytokines in the first days following MI. However, by day 7, there was a significant reduction in the levels of CD45+/Cd11b+ and CD45+/CD11b+/CD86+ cells with HDAC inhibition. Remarkably, HDAC inhibition resulted in the dramatic increase in the recruitment of CD45+/CD11b+/CD206+ alternatively activated macrophages as early as 1 day which remained significantly elevated until 5 days post-MI. qRT-PCR revealed that HDAC inhibitor treatment shifts the cytokine and chemokine environment towards an M2 phenotype with upregulation of M2 markers at 1 and 5 days post-MI. Importantly, HDAC inhibition correlates with significant preservation of both LV ejection fraction and end-diastolic volume and is associated with a significant increase in micro-vessel density in the border zone at 14 days post-MI. CONCLUSION: Inhibition of HDAC activity result in the early recruitment of reparative CD45+/CD11b+/CD206+ macrophages in the post-MI heart and correlates with improved ventricular function and remodeling. This work identifies a very promising therapeutic opportunity to manage macrophage phenotype and enhance resolution of inflammation in the post-MI heart.

Asunto(s)

Histona Desacetilasa 1/genética , Inhibidores de Histona Desacetilasas/administración & dosificación , Inflamación/tratamiento farmacológico , Infarto del Miocardio/tratamiento farmacológico , Cicatrización de Heridas/genética , Animales , Antígeno B7-2/metabolismo , Antígeno CD11b/metabolismo , Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Corazón/crecimiento & desarrollo , Corazón/fisiopatología , Histona Desacetilasa 1/antagonistas & inhibidores , Humanos , Inflamación/genética , Inflamación/fisiopatología , Antígenos Comunes de Leucocito/metabolismo , Leucocitos/metabolismo , Macrófagos/metabolismo , Ratones , Monocitos/efectos de los fármacos , Infarto del Miocardio/genética , Infarto del Miocardio/fisiopatología , Neovascularización Fisiológica/genética , Remodelación Ventricular/efectos de los fármacos , Remodelación Ventricular/genética , Cicatrización de Heridas/efectos de los fármacos

RESUMEN

Myocardial infarctions (MIs) cause the loss of myocytes due to lack of sufficient oxygenation and latent revascularization. Although the administration of histone deacetylase (HDAC) inhibitors reduces the size of infarctions and improves cardiac physiology in small-animal models of MI injury, the cellular targets of the HDACs, which the drugs inhibit, are largely unspecified. Here, we show that WNT-inducible secreted protein-1 (Wisp-1), a matricellular protein that promotes angiogenesis in cancers as well as cell survival in isolated cardiac myocytes and neurons, is a target of HDACs. Further, Wisp-1 transcription is regulated by HDACs and can be modified by the HDAC inhibitor, suberanilohydroxamic acid (SAHA/vorinostat), after MI injury. We observe that, at 7 days after MI, Wisp-1 is elevated 3-fold greater in the border zone of infarction in mice that experience an MI injury and are injected daily with SAHA, relative to MI alone. Additionally, human coronary artery endothelial cells (HCAECs) produce WISP-1 and are responsive to autocrine WISP-1-mediated signaling, which functionally promotes their proangiogenic behavior. Altering endogenous expression of WISP-1 in HCAECs directly impacts their network density in vitro. Therapeutic interventions after a heart attack define the extent of infarct injury, cell survival, and overall prognosis. Our studies shown here identify a potentially novel cardiac angiokine, Wisp-1, that may contribute to beneficial post-MI treatment modalities.

Asunto(s)

Proteínas CCN de Señalización Intercelular/metabolismo , Vasos Coronarios/metabolismo , Histona Desacetilasas/metabolismo , Infarto del Miocardio/patología , Proteínas Proto-Oncogénicas/metabolismo , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Vasos Coronarios/citología , Vasos Coronarios/efectos de los fármacos , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Histona Desacetilasas/uso terapéutico , Humanos , Masculino , Ratones , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/etiología , Miocardio/citología , Miocardio/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Cultivo Primario de Células , Ratas , Ratas Sprague-Dawley , Vorinostat/farmacología , Vorinostat/uso terapéutico

RESUMEN

RATIONALE: Recent evidence indicates that histone deacetylase enzymes (HDACs) contribute to ischemia reperfusion (I/R) injury, and pan-HDAC inhibitors have been shown to be cardioprotective when administered either before an ischemic insult or during reperfusion. We have shown previously that selective inhibition of class I HDACs provides superior cardioprotection when compared to pan-HDAC inhibition in a pretreatment model, but selective class I HDAC inhibition has not been tested during reperfusion, and specific targets of class I HDACs in I/R injury have not been identified. OBJECTIVE: We hypothesized that selective inhibition of class I HDACs with the drug MS-275 (entinostat) during reperfusion would improve recovery from I/R injury in the first hour of reperfusion. METHODS AND RESULTS: Hearts from male Sprague-Dawley rats were subjected to ex vivo I/R injury±MS-275 class I HDAC inhibition during reperfusion alone. MS-275 significantly attenuated I/R injury, as indicated by improved LV function and tissue viability at the end of reperfusion. Unexpectedly, we observed that HDAC1 is present in the mitochondria of cardiac myocytes, but not fibroblasts or endothelial cells. We then designed mitochondria-restricted and mitochondria-excluded HDAC inhibitors, and tested both in our ex vivo I/R model. The selective inhibition of mitochondrial HDAC1 attenuated I/R injury to the same extent as MS-275, whereas the mitochondrial-excluded inhibitor did not. Further assays demonstrated that these effects are attributable to a decrease in SDHA activity and subsequent metabolic ROS production in reperfusion. CONCLUSIONS: We demonstrate for the first time that HDAC1 is present within the mitochondria of cardiac myocytes, and mitochondrial HDAC1 contributes significantly to I/R injury within the first hour of reperfusion.

Asunto(s)

Mitocondrias/enzimología , Daño por Reperfusión Miocárdica/enzimología , Miocitos Cardíacos/enzimología , Animales , Supervivencia Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Histona Desacetilasa 1/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Masculino , Mitocondrias/efectos de los fármacos , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/fisiopatología , Miocardio/enzimología , Miocardio/patología , Miocitos Cardíacos/patología , Consumo de Oxígeno/efectos de los fármacos , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Succinato Deshidrogenasa/metabolismo , Función Ventricular/efectos de los fármacos

RESUMEN

Recent progress in human organoids has provided 3D tissue systems to model human development, diseases, as well as develop cell delivery systems for regenerative therapies. While direct differentiation of human embryoid bodies holds great promise for cardiac organoid production, intramyocardial cell organization during heart development provides biological foundation to fabricate human cardiac organoids with defined cell types. Inspired by the intramyocardial organization events in coronary vasculogenesis, where a diverse, yet defined, mixture of cardiac cell types self-organizes into functional myocardium in the absence of blood flow, we have developed a defined method to produce scaffold-free human cardiac organoids that structurally and functionally resembled the lumenized vascular network in the developing myocardium, supported hiPSC-CM development and possessed fundamental cardiac tissue-level functions. In particular, this development-driven strategy offers a robust, tunable system to examine the contributions of individual cell types, matrix materials and additional factors for developmental insight, biomimetic matrix composition to advance biomaterial design, tissue/organ-level drug screening, and cell therapy for heart repair.

Asunto(s)

Biomimética/métodos , Corazón/embriología , Organoides/embriología , Tejido Adiposo/citología , Matriz Extracelular/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Organoides/citología , Fenotipo , Células Madre/citología , Células Madre/metabolismo

RESUMEN

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide an unlimited cell source to treat cardiovascular diseases, the leading cause of death worldwide. However, current hiPSC-CMs retain an immature phenotype that leads to difficulties for integration with adult myocardium after transplantation. To address this, we recently utilized electrically conductive silicon nanowires (e-SiNWs) to facilitate self-assembly of hiPSC-CMs to form nanowired hiPSC cardiac spheroids. Our previous results showed addition of e-SiNWs effectively enhanced the functions of the cardiac spheroids and improved the cellular maturation of hiPSC-CMs. Here, we examined two important factors that can affect functions of the nanowired hiPSC cardiac spheroids: (1) cell number per spheroid (i.e., size of the spheroids), and (2) the electrical conductivity of the e-SiNWs. To examine the first factor, we prepared hiPSC cardiac spheroids with four different sizes by varying cell number per spheroid (∼0.5k, ∼1k, ∼3k, ∼7k cells/spheroid). Spheroids with ∼3k cells/spheroid was found to maximize the beneficial effects of the 3D spheroid microenvironment. This result was explained with a semi-quantitative theory that considers two competing factors: 1) the improved 3D cell-cell adhesion, and 2) the reduced oxygen supply to the center of spheroids with the increase of cell number. Also, the critical role of electrical conductivity of silicon nanowires has been confirmed in improving tissue function of hiPSC cardiac spheroids. These results lay down a solid foundation to develop suitable nanowired hiPSC cardiac spheroids as an innovative cell delivery system to treat cardiovascular diseases. STATEMENT OF SIGNIFICANCE: Cardiovascular disease is the leading cause of death and disability worldwide. Due to the limited regenerative capacity of adult human hearts, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have received significant attention because they provide a patient specific cell source to regenerate damaged hearts. Despite the progress, current human hiPSC-CMs retain an immature phenotype that leads to difficulties for integration with adult myocardium after transplantation. To address this, we recently utilized electrically conductive silicon nanowires (e-SiNWs) to facilitate self-assembly of hiPSC-CMs to form nanowired hiPSC cardiac spheroids. Our previous results showed addition of e-SiNWs effectively enhanced the functions of the cardiac spheroids and improved the cellular maturation of hiPSC-CMs. In this manuscript, we examined the effects of two important factors on the functions of nanowired hiPSC cardiac spheroids: (1) cell number per spheroid (i.e., size of the spheroids), and (2) the electrical conductivity of the e-SiNWs. The results from these studies will allow for the development of suitable nanowired hiPSC cardiac spheroids to effectively deliver hiPSC-CMs for heart repair.

Asunto(s)

Conductividad Eléctrica , Miocitos Cardíacos/citología , Nanocables/química , Silicio/química , Esferoides Celulares/citología , Recuento de Células , Tamaño de la Célula , Técnica del Anticuerpo Fluorescente , Humanos , Células Madre Pluripotentes Inducidas/citología , Nanocables/ultraestructura , Esferoides Celulares/metabolismo , Esferoides Celulares/ultraestructura

RESUMEN

The advancement of human induced pluripotent stem-cell-derived cardiomyocyte (hiPSC-CM) technology has shown promising potential to provide a patient-specific, regenerative cell therapy strategy to treat cardiovascular disease. Despite the progress, the unspecific, underdeveloped phenotype of hiPSC-CMs has shown arrhythmogenic risk and limited functional improvements after transplantation. To address this, tissue engineering strategies have utilized both exogenous and endogenous stimuli to accelerate the development of hiPSC-CMs. Exogenous electrical stimulation provides a biomimetic pacemaker-like stimuli that has been shown to advance the electrical properties of tissue engineered cardiac constructs. Recently, we demonstrated that the incorporation of electrically conductive silicon nanowires to hiPSC cardiac spheroids led to advanced structural and functional development of hiPSC-CMs by improving the endogenous electrical microenvironment. Here, we reasoned that the enhanced endogenous electrical microenvironment of nanowired hiPSC cardiac spheroids would synergize with exogenous electrical stimulation to further advance the functional development of nanowired hiPSC cardiac spheroids. For the first time, we report that the combination of nanowires and electrical stimulation enhanced cell-cell junction formation, improved development of contractile machinery, and led to a significant decrease in the spontaneous beat rate of hiPSC cardiac spheroids. The advancements made here address critical challenges for the use of hiPSC-CMs in cardiac developmental and translational research and provide an advanced cell delivery vehicle for the next generation of cardiac repair.

Asunto(s)

Estimulación Eléctrica , Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/citología , Nanocables , Diferenciación Celular , Células Cultivadas , Humanos , Silicio

RESUMEN

Histone deacetylases (HDACs) play integral roles in many cardiovascular biological processes ranging from transcriptional and translational regulation to protein stabilization and localization. There are 18 known HDACs categorized into 4 classes that can differ on the basis of substrate targets, subcellular localization, and regulatory binding partners. HDACs are classically known for their ability to remove acetyl groups from histone and nonhistone proteins that have lysine residues. However, despite their nomenclature and classical functions, discoveries from many research groups over the past decade have suggested that nondeacetylase roles exist for class IIa HDACs. This is not surprising given that class IIa HDACs have, for example, relatively poor deacetylase capabilities and are often shuttled in and out of nuclei upon specific pathological and nonpathological cardiac events. This review aims to consolidate and elucidate putative nondeacetylase roles for class IIa HDACs and, where possible, highlight studies that provide evidence for their noncanonical roles, especially in the context of cardiovascular maladies. There has been great interest recently in exploring the pharmacological regulators of HDACs for use in therapeutic interventions for treating cardiovascular diseases and inflammation. Thus it is of interest to earnestly consider nonenzymatic and or nondeacetylase roles of HDACs that might be key in potentiating or abrogating pathologies. These noncanonical HDAC functions may possibly yield new mechanisms and targets for drug discovery.

Asunto(s)

Enfermedades Cardiovasculares/enzimología , Histona Desacetilasas/metabolismo , Animales , Fármacos Cardiovasculares/uso terapéutico , Enfermedades Cardiovasculares/tratamiento farmacológico , Enfermedades Cardiovasculares/genética , Epigénesis Genética , Inhibidores de Histona Desacetilasas/uso terapéutico , Histona Desacetilasas/química , Humanos , Conformación Proteica , Transducción de Señal , Relación Estructura-Actividad

RESUMEN

Class IIa histone deacetylases (HDACs) are very important for tissue specific gene regulation in development and pathology. Because class IIa HDAC catalytic activity is low, their exact molecular roles have not been fully elucidated. Studies have suggested that class IIa HDACs may serve as a scaffold to recruit the catalytically active class I HDAC complexes to their substrate. Here we directly address whether the class IIa HDAC, HDAC5 may function as a scaffold to recruit co-repressor complexes to promoters. We examined two well-characterized cardiac promoters, the sodium calcium exchanger (Ncx1) and the brain natriuretic peptide (Bnp) whose hypertrophic upregulation is mediated by both class I and IIa HDACs. Selective inhibition of class IIa HDACs did not prevent adrenergic stimulated Ncx1 upregulation, however HDAC5 knockout prevented pressure overload induced Ncx1 upregulation. Using the HDAC5((-/-)) mouse we show that HDAC5 is required for the interaction of the HDAC1/2/Sin3a co-repressor complexes with the Nkx2.5 and YY1 transcription factors and critical for recruitment of the HDAC1/Sin3a co-repressor complex to either the Ncx1 or Bnp promoter. Our novel findings support a non-canonical role of class IIa HDACs in the scaffolding of transcriptional regulatory complexes, which may be relevant for therapeutic intervention for pathologies.

Asunto(s)

Regulación de la Expresión Génica/genética , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Péptido Natriurético Encefálico/genética , Intercambiador de Sodio-Calcio/genética , Animales , Gatos , Células Cultivadas , Corazón/crecimiento & desarrollo , Histona Desacetilasa 1/genética , Histona Desacetilasa 1/metabolismo , Proteína Homeótica Nkx-2.5/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Péptido Natriurético Encefálico/metabolismo , Regiones Promotoras Genéticas/genética , Intercambiador de Sodio-Calcio/metabolismo , Transcripción Genética/genética , Activación Transcripcional , Factor de Transcripción YY1/metabolismo

RESUMEN

BACKGROUND: MicroRNAs (miRNAs) and histone deacetylases (HDACs) serve a significant role in the pathogenesis of a variety of cardiovascular diseases. The transcriptional regulation of miRNAs is poorly understood in cardiac hypertrophy. We investigated whether the expression of miR-133a is epigenetically regulated by class I and IIb HDACs during hypertrophic remodeling. METHODS AND RESULTS: Transverse aortic constriction (TAC) was performed in CD1 mice to induce pressure overload hypertrophy. Mice were treated with class I and IIb HDAC inhibitor (HDACi) via drinking water for 2 and 4 weeks post TAC. miRNA expression was determined by real-time polymerase chain reaction. Echocardiography was performed at baseline and post TAC end points for structural and functional assessment. Chromatin immunoprecipitation was used to identify HDACs and transcription factors associated with miR-133a promoter. miR-133a expression was downregulated by 0.7- and 0.5-fold at 2 and 4 weeks post TAC, respectively, when compared with vehicle control (P<0.05). HDAC inhibition prevented this significant decrease 2 weeks post TAC and maintained miR-133a expression near vehicle control levels, which coincided with (1) a decrease in connective tissue growth factor expression, (2) a reduction in cardiac fibrosis and left atrium diameter (marker of end-diastolic pressure), suggesting an improvement in diastolic function. Chromatin immunoprecipitation analysis revealed that HDAC1 and HDAC2 are present on the miR-133a enhancer regions. CONCLUSIONS: The results reveal that HDACs play a role in the regulation of pressure overload-induced miR-133a downregulation. This work is the first to provide insight into an epigenetic-miRNA regulatory pathway in pressure overload-induced cardiac fibrosis.

Asunto(s)

Cardiomegalia/metabolismo , Fibroblastos/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , MicroARNs/metabolismo , Animales , Cardiomegalia/etiología , Cardiomegalia/patología , Técnicas de Cultivo de Célula , Modelos Animales de Enfermedad , Fibroblastos/metabolismo , Histona Desacetilasas/metabolismo , Humanos , Ratones , Vorinostat

RESUMEN

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

Asunto(s)

Preparación de Corazón Aislado/métodos , Daño por Reperfusión Miocárdica/etiología , Daño por Reperfusión Miocárdica/patología , Animales , Cateterismo/métodos , Corazón/fisiopatología , Precondicionamiento Isquémico/métodos , Masculino , Ratas