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1.
Front Pediatr ; 12: 1366953, 2024.
Article in English | MEDLINE | ID: mdl-38745831

ABSTRACT

Objectives: Chest pain is a common chief complaint in pediatric emergency departments (EDs). Coronavirus disease-2019 (COVID-19) has been shown to increase the risk of cardiac disease. It remains unclear how COVID-19 changed how pediatric emergency clinicians approach patients presenting with chest pain. The goal of this study was to characterize the diagnostic testing for chest pain in a pediatric ED before and during the COVID-19 pandemic. Methods: This was a retrospective study of children between the ages of 2-17 years presenting to a pediatric ED from 1/1/2018-2/29/2020 (Pre-COVID-19) and 3/1/2020-4/30/2022 (COVID-19) with chest pain. We excluded patients with a previous history of cardiac disease. Results: Of the 10,721 encounters during the study period, 5,692 occurred before and 5,029 during COVID-19. Patient demographics showed minor differences by age, weight, race and ethnicity. ED encounters for chest pain consisted of an average of 18% more imaging studies during COVID-19, including 14% more EKGs and 11% more chest x-rays, with no difference in the number of echocardiograms. Compared to Pre-COVID-19, 100% more diagnostic tests were ordered during COVID-19, including cardiac markers Troponin I (p < 0.001) and BNP (p < 0.001). During COVID-19, 1.1% of patients had a cardiac etiology of chest pain compared with 0.7% before COVID-19 (p = 0.03). Conclusions: During COVID-19, pediatric patients with chest pain underwent more diagnostic testing compared to Pre-COVID-19. This may be due to higher patient acuity, emergence of multisystem inflammatory syndrome in children (MIS-C) that necessitated more extensive testing and possible changes in ED clinician behavior during COVID-19.

2.
Nat Med ; 29(2): 401-411, 2023 02.
Article in English | MEDLINE | ID: mdl-36797478

ABSTRACT

The most common form of genetic heart disease is hypertrophic cardiomyopathy (HCM), which is caused by variants in cardiac sarcomeric genes and leads to abnormal heart muscle thickening. Complications of HCM include heart failure, arrhythmia and sudden cardiac death. The dominant-negative c.1208G>A (p.R403Q) pathogenic variant (PV) in ß-myosin (MYH7) is a common and well-studied PV that leads to increased cardiac contractility and HCM onset. In this study we identify an adenine base editor and single-guide RNA system that can efficiently correct this human PV with minimal bystander editing and off-target editing at selected sites. We show that delivery of base editing components rescues pathological manifestations of HCM in induced pluripotent stem cell cardiomyocytes derived from patients with HCM and in a humanized mouse model of HCM. Our findings demonstrate the potential of base editing to treat inherited cardiac diseases and prompt the further development of adenine base editor-based therapies to correct monogenic variants causing cardiac disease.


Subject(s)
Cardiomyopathy, Hypertrophic , Myocytes, Cardiac , Humans , Animals , Mice , Gene Editing , Myocardium , Arrhythmias, Cardiac , Mutation
3.
Nat Commun ; 12(1): 5270, 2021 09 06.
Article in English | MEDLINE | ID: mdl-34489413

ABSTRACT

Following injury, cells in regenerative tissues have the ability to regrow. The mechanisms whereby regenerating cells adapt to injury-induced stress conditions and activate the regenerative program remain to be defined. Here, using the mammalian neonatal heart regeneration model, we show that Nrf1, a stress-responsive transcription factor encoded by the Nuclear Factor Erythroid 2 Like 1 (Nfe2l1) gene, is activated in regenerating cardiomyocytes. Genetic deletion of Nrf1 prevented regenerating cardiomyocytes from activating a transcriptional program required for heart regeneration. Conversely, Nrf1 overexpression protected the adult mouse heart from ischemia/reperfusion (I/R) injury. Nrf1 also protected human induced pluripotent stem cell-derived cardiomyocytes from doxorubicin-induced cardiotoxicity and other cardiotoxins. The protective function of Nrf1 is mediated by a dual stress response mechanism involving activation of the proteasome and redox balance. Our findings reveal that the adaptive stress response mechanism mediated by Nrf1 is required for neonatal heart regeneration and confers cardioprotection in the adult heart.


Subject(s)
Heart/physiology , Myocardial Reperfusion Injury/metabolism , NF-E2-Related Factor 1/metabolism , Animals , Animals, Newborn , Cell Differentiation/drug effects , Cell Differentiation/physiology , Doxorubicin/pharmacology , Female , Heme Oxygenase (Decyclizing)/genetics , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/drug effects , Male , Mice, Knockout , Mice, Transgenic , Myocardial Reperfusion Injury/pathology , Myocytes, Cardiac/physiology , NF-E2-Related Factor 1/genetics , Oxidation-Reduction , Proteostasis , Rats, Sprague-Dawley , Regeneration
4.
Mol Ther Methods Clin Dev ; 22: 122-132, 2021 Sep 10.
Article in English | MEDLINE | ID: mdl-34485599

ABSTRACT

Duchenne muscular dystrophy (DMD), caused by mutations in the X-linked dystrophin gene, is a lethal neuromuscular disease. Correction of DMD mutations in animal models has been achieved by CRISPR/Cas9 genome editing using Streptococcus pyogenes Cas9 (SpCas9) delivered by adeno-associated virus (AAV). However, due to the limited viral packaging capacity of AAV, two AAV vectors are required to deliver the SpCas9 nuclease and its single guide RNA (sgRNA), impeding its therapeutic application. We devised an efficient single-cut gene-editing method using a compact Staphylococcus aureus Cas9 (SaCas9) to restore the open reading frame of exon 51, the most commonly affected out-of-frame exon in DMD. Editing of exon 51 in cardiomyocytes derived from human induced pluripotent stem cells revealed a strong preference for exon reframing via a two-nucleotide deletion. We adapted this system to express SaCas9 and sgRNA from a single AAV9 vector. Systemic delivery of this All-In-One AAV9 system restored dystrophin expression and improved muscle contractility in a mouse model of DMD with exon 50 deletion. These findings demonstrate the effectiveness of CRISPR/SaCas9 delivered by a consolidated AAV delivery system in the correction of DMD in vivo, representing a promising therapeutic approach to correct the genetic causes of DMD.

6.
Elife ; 82019 10 08.
Article in English | MEDLINE | ID: mdl-31591966

ABSTRACT

A fundamental goal in the biological sciences is to determine how individual cells with varied gene expression profiles and diverse functional characteristics contribute to development, physiology, and disease. Here, we report a novel strategy to assess gene expression and cell physiology in single living cells. Our approach utilizes fluorescently labeled mRNA-specific anti-sense RNA probes and dsRNA-binding protein to identify the expression of specific genes in real-time at single-cell resolution via FRET. We use this technology to identify distinct myocardial subpopulations expressing the structural proteins myosin heavy chain α and myosin light chain 2a in real-time during early differentiation of human pluripotent stem cells. We combine this live-cell gene expression analysis with detailed physiologic phenotyping to capture the functional evolution of these early myocardial subpopulations during lineage specification and diversification. This live-cell mRNA imaging approach will have wide ranging application wherever heterogeneity plays an important biological role.


Subject(s)
Fluorescence Resonance Energy Transfer/methods , Gene Expression Profiling/methods , Intravital Microscopy/methods , Single-Cell Analysis/methods , Cell Differentiation , Humans , Myocytes, Cardiac/physiology , Pluripotent Stem Cells/physiology , Staining and Labeling/methods
7.
Trends Cell Biol ; 27(5): 352-364, 2017 05.
Article in English | MEDLINE | ID: mdl-28007424

ABSTRACT

The advent of human pluripotent stem cell (hPSC) biology has opened unprecedented opportunities for the use of tissue engineering to generate human cardiac tissue for in vitro study. Engineering cardiac constructs that recapitulate human development and disease requires faithful recreation of the cardiac niche in vitro. Here we discuss recent progress in translating the in vivo cardiac microenvironment into PSC models of the human heart. We review three key physiologic features required to recreate the cardiac niche and facilitate normal cardiac differentiation and maturation: the biochemical, biophysical, and bioelectrical signaling cues. Finally, we discuss key barriers that must be overcome to fulfill the promise of stem cell biology in preclinical applications and ultimately in clinical practice.


Subject(s)
Cellular Microenvironment , Disease , Models, Biological , Physiological Phenomena , Pluripotent Stem Cells/cytology , Humans
8.
Cardiovasc Diabetol ; 15: 75, 2016 May 06.
Article in English | MEDLINE | ID: mdl-27153943

ABSTRACT

BACKGROUND: Type-2 diabetics have an increased risk of cardiomyopathy, and heart failure is a major cause of death among these patients. Growing evidence indicates that proinflammatory cytokines may induce the development of insulin resistance, and that anti-inflammatory medications may reverse this process. We investigated the effects of the oral administration of zinc and acetylsalicylic acid, in the form of bis(aspirinato)zinc(II)-complex Zn(ASA)2, on different aspects of cardiac damage in Zucker diabetic fatty (ZDF) rats, an experimental model of type-2 diabetic cardiomyopathy. METHODS: Nondiabetic control (ZL) and ZDF rats were treated orally with vehicle or Zn(ASA)2 for 24 days. At the age of 29-30 weeks, the electrical activities, left-ventricular functional parameters and left-ventricular wall thicknesses were assessed. Nitrotyrosine immunohistochemistry, TUNEL-assay, and hematoxylin-eosin staining were performed. The protein expression of the insulin-receptor and PI3K/AKT pathway were quantified by Western blot. RESULTS: Zn(ASA)2-treatment significantly decreased plasma glucose concentration in ZDF rats (39.0 ± 3.6 vs 49.4 ± 2.8 mM, P < 0.05) while serum insulin-levels were similar among the groups. Data from cardiac catheterization showed that Zn(ASA)2 normalized the increased left-ventricular diastolic stiffness (end-diastolic pressure-volume relationship: 0.064 ± 0.008 vs 0.084 ± 0.014 mmHg/µl; end-diastolic pressure: 6.5 ± 0.6 vs 7.9 ± 0.7 mmHg, P < 0.05). Furthermore, ECG-recordings revealed a restoration of prolonged QT-intervals (63 ± 3 vs 83 ± 4 ms, P < 0.05) with Zn(ASA)2. Left-ventricular wall thickness, assessed by echocardiography, did not differ among the groups. However histological examination revealed an increase in the cardiomyocytes' transverse cross-section area in ZDF compared to the ZL rats, which was significantly decreased after Zn(ASA)2-treatment. Additionally, a significant fibrotic remodeling was observed in the diabetic rats compared to ZL rats, and Zn(ASA)2-administered ZDF rats showed a similar collagen content as ZL animals. In diabetic hearts Zn(ASA)2 significantly decreased DNA-fragmentation, and nitro-oxidative stress, and up-regulated myocardial phosphorylated-AKT/AKT protein expression. Zn(ASA)2 reduced cardiomyocyte death in a cellular model of oxidative stress. Zn(ASA)2 had no effects on altered myocardial CD36, GLUT-4, and PI3K protein expression. CONCLUSIONS: We demonstrated that treatment of type-2 diabetic rats with Zn(ASA)2 reduced plasma glucose-levels and prevented diabetic cardiomyopathy. The increased myocardial AKT activation could, in part, help to explain the cardioprotective effects of Zn(ASA)2. The oral administration of Zn(ASA)2 may have therapeutic potential, aiming to prevent/treat cardiac complications in type-2 diabetic patients.


Subject(s)
Aspirin/pharmacology , Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Type 2/drug therapy , Diabetic Cardiomyopathies/drug therapy , Signal Transduction/drug effects , Zinc/pharmacokinetics , Administration, Oral , Animals , Aspirin/administration & dosage , Aspirin/therapeutic use , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Type 2/blood , Diabetic Cardiomyopathies/metabolism , Heart Ventricles/drug effects , Heart Ventricles/physiopathology , Male , Proto-Oncogene Proteins c-akt/metabolism , Rats , Rats, Zucker , Zinc/administration & dosage
9.
Cell Rep ; 14(7): 1662-1672, 2016 Feb 23.
Article in English | MEDLINE | ID: mdl-26876178

ABSTRACT

A hallmark of cardiac development is the formation of myocardial trabeculations exclusively from the luminal surface of the primitive heart tube. Although a number of genetic defects in the endocardium and cardiac jelly disrupt myocardial trabeculation, the role of cell polarization remains unclear. Here, we demonstrate that atypical protein kinase C iota (Prkci) and its interacting partners are localized primarily to the luminal side of myocardial cells of early murine embryonic hearts. A subset of these cells undergoes polarized cell division with the cell division plane perpendicular to the heart's lumen. Disruption of the cell polarity complex by targeted gene mutations results in aberrant mitotic spindle alignment, loss of polarized cardiomyocyte division, and loss of normal myocardial trabeculation. Collectively, these results suggest that, in response to inductive signals, Prkci and its downstream partners direct polarized cell division of luminal myocardial cells to drive trabeculation in the nascent heart.


Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Cell Division/genetics , Isoenzymes/genetics , Myocardium/metabolism , Myocytes, Cardiac/metabolism , Organogenesis/genetics , Protein Kinase C/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Antigens/genetics , Antigens/metabolism , Cell Polarity , Embryo, Mammalian , Endocardium/embryology , Endocardium/metabolism , Gene Expression Regulation, Developmental , Histones/genetics , Histones/metabolism , Isoenzymes/metabolism , Mice , Myocytes, Cardiac/ultrastructure , Protein Binding , Protein Kinase C/metabolism , Signal Transduction , Spindle Apparatus/metabolism , Spindle Apparatus/ultrastructure , Troponin T/genetics , Troponin T/metabolism
10.
J Physiol Sci ; 66(2): 113-25, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26497333

ABSTRACT

We recently demonstrated that the pre-treatment of rats with zinc and acetylsalicylic acid complex in the form of bis(aspirinato)zinc(II) [Zn(ASA)2] is superior to acetylsalicylic acid in protecting the heart from acute myocardial ischemia. Herein, we hypothesized that Zn(ASA)2 treatment after the onset of an acute myocardial injury could protect the heart. The rats were treated with a vehicle or Zn(ASA)2 after an isoproterenol injection. Isoproterenol-induced cardiac damage [inflammatory infiltration into myocardial tissue, DNA-strand breakage evidenced by TUNEL-assay, increased 11-dehydro thromboxane (TX)B2-levels, elevated ST-segment, widened QRS complex and prolonged QT-interval] was prevented by the Zn(ASA)2 treatment. In isoproterenol-treated rats, load-independent left ventricular contractility parameters were significantly improved after Zn(ASA)2. Furthermore, Zn(ASA)2 significantly increased the myocardial mRNA-expression of superoxide dismutase-1, glutathione peroxidase-4 and decreased the level of Na(+)/K(+)/ATPase. Postconditioning with Zn(ASA)2 protects the heart from acute myocardial ischemia. Its mechanisms of action might involve inhibition of pro-inflammatory prostanoids and upregulation of antioxidant enzymes.


Subject(s)
Antioxidants/metabolism , Aspirin/administration & dosage , Heart Injuries/drug therapy , Heart Ventricles/drug effects , Myocardial Infarction/drug therapy , Up-Regulation/drug effects , Zinc/administration & dosage , Animals , Glutathione Peroxidase/metabolism , Heart Injuries/metabolism , Heart Ventricles/metabolism , Male , Myocardial Infarction/metabolism , Myocardium/metabolism , Phospholipid Hydroperoxide Glutathione Peroxidase , Rats , Rats, Sprague-Dawley , Sodium-Potassium-Exchanging ATPase/metabolism , Superoxide Dismutase/metabolism , Superoxide Dismutase-1
11.
Exp Biol Med (Maywood) ; 240(9): 1247-55, 2015 Sep.
Article in English | MEDLINE | ID: mdl-25670850

ABSTRACT

The pathophysiology of ischemic myocardial injury involves cellular events, reactive oxygen species, and an inflammatory reaction cascade. The zinc complex of acetylsalicylic acid (Zn(ASA)2) has been found to possess higher anti-inflammatory and lower ulcerogenic activities than acetylsalicylic acid (ASA). Herein, we studied the effects of both ASA and Zn(ASA)2 against acute myocardial ischemia. Rats were pretreated with ASA (75 mg/kg) or Zn(ASA)2 (100 mg/kg) orally for five consecutive days. Isoproterenol (85 mg/kg, subcutaneously [s.c.]) was applied to produce myocardial infarction. After 17-22 h, animals were anesthetized with sodium pentobarbital (60 mg/kg, intraperitoneally [i.p.]) and both electrical and mechanical parameters of cardiac function were evaluated in vivo. Myocardial histological and gene expression analyses were performed. In isoproterenol-treated rats, Zn(ASA)2 treatment normalized significantly impaired left-ventricular contractility index (Emax 2.6 ± 0.7 mmHg/µL vs. 4.6 ± 0.5 mmHg/µL, P < 0.05), increased stroke volume (30 ± 3 µL vs. 50 ± 6 µL, P < 0.05), decreased systemic vascular resistance (7.2 ± 0.7 mmHg/min/mL vs. 4.2 ± 0.5 mmHg/min/mL, P < 0.05) and reduced inflammatory infiltrate into the myocardial tissues. ECG revealed a restoration of elevated ST-segment (0.21 ± 0.03 mV vs. 0.09 ± 0.02 mV, P < 0.05) and prolonged QT-interval (79.2 ± 3.2 ms vs. 69.5 ± 2.5 ms, P < 0.05) by Zn(ASA)2. ASA treatment did not result in an improvement of these parameters. Additionally, Zn(ASA)2 significantly increased the mRNA-expression of superoxide dismutase 1 (+73 ± 15%), glutathione peroxidase 4 (+44 ± 12%), and transforming growth factor (TGF)-ß1 (+102 ± 22%). In conclusion, our data demonstrate that oral administration of zinc and ASA in the form of bis(aspirinato)zinc(II) complex is superior to ASA in preventing electrical, mechanical, and histological changes after acute myocardial ischemia. The induction of antioxidant enzymes and the anti-inflammatory cytokine TGF-ß1 may play a pivotal role in the mechanism of action of Zn(ASA)2.


Subject(s)
Aspirin/analogs & derivatives , Coordination Complexes/administration & dosage , Myocardial Ischemia/prevention & control , Animals , Anti-Inflammatory Agents, Non-Steroidal/administration & dosage , Aspirin/administration & dosage , Electrocardiography , Gene Expression Regulation/drug effects , Hemodynamics/drug effects , Isoproterenol/toxicity , Male , Myocardial Contraction/drug effects , Myocardial Infarction/chemically induced , Myocardial Infarction/complications , Myocardial Infarction/pathology , Myocardial Ischemia/etiology , Myocardial Ischemia/physiopathology , Rats , Rats, Sprague-Dawley , Troponin T/blood , Zinc/administration & dosage
13.
Development ; 140(20): 4165-76, 2013 Oct.
Article in English | MEDLINE | ID: mdl-24026118

ABSTRACT

In mammals, cardiac development proceeds from the formation of the linear heart tube, through complex looping and septation, all the while increasing in mass to provide the oxygen delivery demands of embryonic growth. The developing heart must orchestrate regional differences in cardiomyocyte proliferation to control cardiac morphogenesis. During ventricular wall formation, the compact myocardium proliferates more vigorously than the trabecular myocardium, but the mechanisms controlling such regional differences among cardiomyocyte populations are not understood. Control of definitive cardiomyocyte proliferation is of great importance for application to regenerative cell-based therapies. We have used murine and human pluripotent stem cell systems to demonstrate that, during in vitro cellular differentiation, early ventricular cardiac myocytes display a robust proliferative response to ß-catenin-mediated signaling and conversely accelerate differentiation in response to inhibition of this pathway. Using gain- and loss-of-function murine genetic models, we show that ß-catenin controls ventricular myocyte proliferation during development and the perinatal period. We further demonstrate that the differential activation of the Wnt/ß-catenin signaling pathway accounts for the observed differences in the proliferation rates of the compact versus the trabecular myocardium during normal cardiac development. Collectively, these results provide a mechanistic explanation for the differences in localized proliferation rates of cardiac myocytes and point to a practical method for the generation of the large numbers of stem cell-derived cardiac myocytes necessary for clinical applications.


Subject(s)
Heart Ventricles/cytology , Myocytes, Cardiac/cytology , Pluripotent Stem Cells/metabolism , Wnt Proteins/metabolism , Wnt Signaling Pathway , beta Catenin/metabolism , Animals , Cell Differentiation , Cell Proliferation , Cells, Cultured , Enzyme Activation , Gene Expression Regulation, Developmental , Heart Ventricles/embryology , Humans , Mice , Morphogenesis , Myocytes, Cardiac/metabolism
14.
J Vis Exp ; (73): e50288, 2013 Mar 19.
Article in English | MEDLINE | ID: mdl-23542789

ABSTRACT

Advanced heart failure represents a major unmet clinical challenge, arising from the loss of viable and/or fully functional cardiac muscle cells. Despite optimum drug therapy, heart failure represents a leading cause of mortality and morbidity in the developed world. A major challenge in drug development is the identification of cellular assays that accurately recapitulate normal and diseased human myocardial physiology in vitro. Likewise, the major challenges in regenerative cardiac biology revolve around the identification and isolation of patient-specific cardiac progenitors in clinically relevant quantities. These cells have to then be assembled into functional tissue that resembles the native heart tissue architecture. Microcontact printing allows for the creation of precise micropatterned protein shapes that resemble structural organization of the heart, thus providing geometric cues to control cell adhesion spatially. Herein we describe our approach for the isolation of highly purified myocardial cells from pluripotent stem cells differentiating in vitro, the generation of cell growth surfaces micropatterned with extracellular matrix proteins, and the assembly of the stem cell-derived cardiac muscle cells into anisotropic myocardial tissue.


Subject(s)
Heart/anatomy & histology , Myocardium/cytology , Pluripotent Stem Cells/cytology , Printing/methods , Animals , Cell Adhesion/physiology , Cell Differentiation , Cell Growth Processes , Cell Separation/methods , Extracellular Matrix Proteins/chemistry , Mice , Mice, Transgenic , Printing/instrumentation , Tissue Engineering/methods
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