CRISPR/Cas9-based targeting of fluorescent reporters to human iPSCs to isolate atrial and ventricular-specific cardiomyocytes.
2021; 11 (1): 3026
Pectoral Nerve Blocks Decrease Postoperative Pain and Opioid Use After Pacemaker or Implantable Cardioverter-Defibrillator Placement in Children.
Generating cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) has represented a significant advance in our ability to model cardiac disease. Current differentiation protocols, however, have limited use due to their production of heterogenous cell populations, primarily consisting of ventricular-like CMs. Here we describe the creation of two chamber-specific reporter hiPSC lines by site-directed genomic integration using CRISPR-Cas9 technology. In the MYL2-tdTomato reporter, the red fluorescent tdTomato was inserted upstream of the 3' untranslated region of the Myosin Light Chain 2 (MYL2) gene in order faithfully label hiPSC-derived ventricular-like CMs while avoiding disruption of endogenous gene expression. Similarly, in the SLN-CFP reporter, Cyan Fluorescent Protein (CFP) was integrated downstream of the coding region of the atrial-specific gene, Sarcolipin (SLN). Purification of tdTomato+ and CFP+ CMs using flow cytometry coupled with transcriptional and functional characterization validated these genetic tools for their use in the isolation of bona fide ventricular-like and atrial-like CMs, respectively. Finally, we successfully generated a double reporter system allowing for the isolation of both ventricular and atrial CM subtypes within a single hiPSC line. These tools provide a platform for chamber-specific hiPSC-derived CM purification and analysis in the context of atrial- or ventricular-specific disease and therapeutic opportunities.
View details for DOI 10.1038/s41598-021-81860-x
View details for PubMedID 33542270
Wnt Activation and Reduced Cell-Cell Contact Synergistically Induce Massive Expansion of Functional Human iPSC-Derived Cardiomyocytes.
Cell stem cell
2020; 27 (1): 50–63.e5
BACKGROUND: Pectoral nerve blocks (PECS) can reduce intra-procedural anesthetic requirements and postoperative pain. Little is known on the utility of PECS in reducing pain and narcotic use after pacemaker (PM) or implantable cardioverter defibrillator (ICD) placement in children.OBJECTIVE: To determine whether PECS can decrease postoperative pain and opioid use after PM or ICD placement in children.METHODS: A single center, retrospective review of pediatric patients undergoing transvenous PM or ICD placement between 2015-2020 was performed. Patients with recent cardiothoracic surgery or neurologic/developmental deficits were excluded. Demographics, procedural variables, postoperative pain, and postoperative opioid usage were compared between patients who underwent PECS and those who underwent conventional local anesthetic (CONTROL).RESULTS: A total of 74 patients underwent PM or ICD placement with 20 patients (27%) undergoing PECS. There were no differences between PECS and CONTROL with regard to age, weight, gender, type of device placed, presence of congenital heart disease, type of anesthesia, procedural time or complication rates. Patients who underwent PECS had lower pain scores at 1, 2, 6, 18, and 24-hours compared to CONTROL. PECS patients had a lower mean cumulative pain score [PECS 1.5 (95%-CI 0.8-2.2) vs CONTROL 3.1 (95%-CI 2.7-3.5); P<0.001] and lower total opioid use [PECS 6.0 MME/m2 (95%-CI 3.4-8.6) vs CONTROL 15.0 MME/m2 (95%-CI 11.8-18.2); P=0.001] over the 24-hours post-implant.CONCLUSIONS: Pectoralis nerve blocks reduce postoperative pain scores and lower total opioid usage after ICD or PM placement. PECS should be considered at the time of transvenous device placement in children.
View details for DOI 10.1016/j.hrthm.2020.03.009
View details for PubMedID 32201270
Broad Genetic Testing in a Clinical Setting Uncovers a High Prevalence of Titin Loss-of-Function Variants in Very Early-Onset Atrial Fibrillation.
Circulation. Genomic and precision medicine
Modulating signaling pathways including Wnt and Hippo can induce cardiomyocyte proliferation in vivo. Applying these signaling modulators to human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in vitro can expand CMs modestly (<5-fold). Here, we demonstrate massive expansion of hiPSC-CMs in vitro (i.e., 100- to 250-fold) by glycogen synthase kinase-3β (GSK-3β) inhibition using CHIR99021 and concurrent removal of cell-cell contact. We show that GSK-3β inhibition suppresses CM maturation, while contact removal prevents CMs from cell cycle exit. Remarkably, contact removal enabled 10 to 25 times greater expansion beyond GSK-3β inhibition alone. Mechanistically, persistent CM proliferation required both LEF/TCF activity and AKT phosphorylation but was independent from yes-associated protein (YAP) signaling. Engineered heart tissues from expanded hiPSC-CMs showed comparable contractility to those from unexpanded hiPSC-CMs, demonstrating uncompromised cellular functionality after expansion. In summary, we uncovered a molecular interplay that enables massive hiPSC-CM expansion for large-scale drug screening and tissue engineering applications.
View details for DOI 10.1016/j.stem.2020.06.001
View details for PubMedID 32619518
Outcomes After Aortopulmonary Window for Hypoplastic Pulmonary Arteries and Dual-Supply Collaterals
ANNALS OF THORACIC SURGERY
2019; 108 (3): 820–27
Transcriptomic Profiling of the Developing Cardiac Conduction System at Single-Cell Resolution.
Atrial fibrillation (AF) is the most common sustained arrhythmia, affecting approximately 34 million worldwide. The pathophysiology of AF remains incompletely understood but is clearly complex with multiple underlying genetic, physiologic and environmental factors. Very early-onset AF (vEAF) (defined here as onset <45 years and without significant comorbidities), while rare (only ~0.5-3% of AF cases), is highly heritable, with a greater prevalence of rare variants in genes previously associated with AF. Patients with vEAF, therefore, represent an ideal population for discovering novel genes involved in the underlying genetic basis of AF. Notably, the Framingham study showed that patients with AF without comorbidities have a three-fold higher risk for heart failure. Conversely, several forms of inherited cardiomyopathy have been strongly associated with AF suggestive of a shared etiology.
View details for DOI 10.1161/CIRCGEN.119.002713
View details for PubMedID 31638414
Single cell expression analysis reveals anatomical and cell cycle-dependent transcriptional shifts during heart development.
Development (Cambridge, England)
RATIONALE: The cardiac conduction system (CCS) consists of distinct components including the sinoatrial node (SAN), atrioventricular node (AVN), His bundle, bundle branches (BB) and Purkinje fibers (PF). Despite an essential role for the CCS in heart development and function, the CCS has remained challenging to interrogate due to inherent obstacles including small cell numbers, large cell type heterogeneity, complex anatomy and difficulty in isolation. Single-cell RNA-sequencing (scRNA-seq) allows for genome-wide analysis of gene expression at single-cell resolution.OBJECTIVE: Assess the transcriptional landscape of the entire CCS at single-cell resolution by scRNA-seq within the developing mouse heart.METHODS AND RESULTS: Wild-type, embryonic day 16.5 mouse hearts (n=6 per zone) were harvested and three zones of microdissection were isolated, including: Zone I - SAN region; Zone II - AVN/His region; and Zone III - BB/PF region. Tissue was digested into single cell suspensions, isolated, reverse transcribed and barcoded prior to high-throughput sequencing and bioinformatics analyses. scRNA-seq was performed on over 22,000 cells and all major cell types of the murine heart were successfully captured including bona fide clusters of cells consistent with each major component of the CCS. Unsupervised weighted gene co-expression network analysis led to the discovery of a host of novel CCS genes, a subset of which were validated using fluorescent in situ hybridization as well as whole mount immunolabelling with volume imaging (iDISCO+) in three-dimensions on intact mouse hearts. Further, subcluster analysis unveiled isolation of distinct CCS cell subtypes, including the clinically-relevant but poorly characterized "transitional cells" that bridge the CCS and surrounding myocardium.CONCLUSIONS: Our study represents the first comprehensive assessment of the transcriptional profiles from the entire CCS at single-cell resolution and provides a gene atlas for facilitating future efforts in conduction cell identification, isolation and characterization in the context of development and disease.
View details for DOI 10.1161/CIRCRESAHA.118.314578
View details for PubMedID 31284824
Outcomes After Aortopulmonary Window for Hypoplastic Pulmonary Arteries and Dual-Supply Collaterals.
The Annals of thoracic surgery
The heart is a complex organ composed of multiple cell and tissue types. Cardiac cells from different regions of the growing embryonic heart exhibit distinct patterns of gene expression, which are thought to contribute to heart development and morphogenesis. Single cell RNA sequencing allows genome-wide analysis of gene expression at the single cell level. Here, we analyzed cardiac cells derived from early stage developing hearts by single cell RNA-seq and identified cell cycle gene expression as a major determinant of transcriptional variation. Within cell cycle stage-matched CMs from a given heart chamber, we found that CMs in the G2/M phase downregulated sarcomeric and cytoskeletal markers. We also identified cell location-specific signaling molecules that may influence the proliferation of other nearby cell types. Our data highlight how variations in cell cycle activity selectively promote cardiac chamber growth during development, reveal profound chamber-specific cell cycle-linked transcriptional shifts, and open the way to deeper understanding of pathogenesis of congenital heart disease.
View details for DOI 10.1242/dev.173476
View details for PubMedID 31142541
Swinging beats: transient heart block in cardiac lymphoma
NETHERLANDS HEART JOURNAL
2018; 26 (9): 467–68
Fates Aligned: Origins and Mechanisms of Ventricular Conduction System and Ventricular Wall Development.
BACKGROUND: Our institutional approach to tetralogy of Fallot (TOF) with major aortopulmonary collateral arteries (MAPCAs) emphasizes early unifocalization and complete repair (CR). In the small subset of patients with dual-supply MAPCAs and confluent but hypoplastic central pulmonary arteries (PAs), our surgical approach is early creation of an aortopulmonary window (APW) to promote PA growth. Factors associated with successful progression to CR, and mid-term outcomes have not been assessed.METHODS: Clinical data were reviewed. PA diameters were measured off-line from angiograms prior to APW and on follow-up catheterization >1 month after APW but prior to any additional surgical interventions.RESULTS: From 11/01-3/18, 352 patients with TOF/MAPCAs underwent initial surgery at our center, 40 of whom had a simple APW with or without ligation of MAPCAs as the first procedure (median age 1.4 months). All PA diameters increased significantly on follow-up angiography. Ultimately, 35 patients underwent CR after APW. Nine of these patients (26%) underwent intermediate palliative operation between 5 and 39 months (median 8 months). There were no early deaths. The cumulative incidence of CR was 65% 1 year post-APW and 87% at 3 years. Repaired patients were followed for a median of 4.2 years after repair; the median PA:aortic pressure ratio was 0.39 (0.22-0.74).CONCLUSIONS: Most patients with TOF/MAPCAs and hypoplastic but normally arborizing PAs and dual-supply MAPCAs are able to undergo CR with low right ventricular pressure after APW early in life. Long-term outcomes were good, with acceptable PA pressures in most patients.
View details for PubMedID 30980823
Neonatal beta Cell Development in Mice and Humans Is Regulated by Calcineurin/NFAT
2012; 23 (1): 21-34
The cardiac conduction system is a network of distinct cell types necessary for the coordinated contraction of the cardiac chambers. The distal portion, known as the ventricular conduction system, allows for the rapid transmission of impulses from the atrio-ventricular node to the ventricular myocardium and plays a central role in cardiac function as well as disease when perturbed. Notably, its patterning during embryogenesis is intimately linked to that of ventricular wall formation, including trabeculation and compaction. Here, we review our current understanding of the underlying mechanisms responsible for the development and maturation of these interdependent processes.
View details for PubMedID 29594502
Deconstructing Pancreas Developmental Biology
COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY
2012; 4 (6)
Little is known about the mechanisms governing neonatal growth and maturation of organs. Here we demonstrate that calcineurin/Nuclear Factor of Activated T cells (Cn/NFAT) signaling regulates neonatal pancreatic development in mouse and human islets. Inactivation of calcineurin b1 (Cnb1) in mouse islets impaired dense core granule biogenesis, decreased insulin secretion, and reduced cell proliferation and mass, culminating in lethal diabetes. Pancreatic β cells lacking Cnb1 failed to express genes revealed to be direct NFAT targets required for replication, insulin storage, and secretion. In contrast, glucokinase activation stimulated Cn-dependent expression of these genes. Calcineurin inhibitors, such as tacrolimus, used for human immunosuppression, induce diabetes. Tacrolimus exposure reduced Cn/NFAT-dependent expression of factors essential for insulin dense core granule formation and secretion and neonatal β cell proliferation, consistent with our genetic studies. Discovery of conserved pathways regulating β cell maturation and proliferation suggests new strategies for controlling β cell growth or replacement in human islet diseases.
View details for DOI 10.1016/j.devcel.2012.05.014
View details for Web of Science ID 000306583800007
View details for PubMedID 22814600
View details for PubMedCentralID PMC3587727
HTP-3 links DSB formation with homolog pairing and crossing over during C. elegans meiosis
2008; 14 (2): 263-274
The relentless nature and increasing prevalence of human pancreatic diseases, in particular, diabetes mellitus and adenocarcinoma, has motivated further understanding of pancreas organogenesis. The pancreas is a multifunctional organ whose epithelial cells govern a diversity of physiologically vital endocrine and exocrine functions. The mechanisms governing the birth, differentiation, morphogenesis, growth, maturation, and maintenance of the endocrine and exocrine components in the pancreas have been discovered recently with increasing tempo. This includes recent studies unveiling mechanisms permitting unexpected flexibility in the developmental potential of immature and mature pancreatic cell subsets, including the ability to interconvert fates. In this article, we describe how classical cell biology, genetic analysis, lineage tracing, and embryological investigations are being complemented by powerful modern methods including epigenetic analysis, time-lapse imaging, and flow cytometry-based cell purification to dissect fundamental processes of pancreas development.
View details for DOI 10.1101/cshperspect.a012401
View details for Web of Science ID 000308028500015
View details for PubMedID 22587935
View details for PubMedCentralID PMC3367550
Social learning and innovation are positively correlated in pigeons (Columba livia)
2007; 10 (2): 259-266
Repair of the programmed meiotic double-strand breaks (DSBs) that initiate recombination must be coordinated with homolog pairing to generate crossovers capable of directing chromosome segregation. Chromosome pairing and synapsis proceed independently of recombination in worms and flies, suggesting a paradoxical lack of coregulation. Here, we find that the meiotic axis component HTP-3 links DSB formation with homolog pairing and synapsis. HTP-3 forms complexes with the DSB repair components MRE-11/RAD-50 and the meiosis-specific axis component HIM-3. Loss of htp-3 or mre-11 recapitulates meiotic phenotypes consistent with a failure to generate DSBs, suggesting that HTP-3 associates with MRE-11/RAD-50 in a complex required for meiotic DSB formation. Loss of HTP-3 eliminates HIM-3 localization to axes and HIM-3-dependent homolog alignment, synapsis, and crossing over. Our study reveals a mechanism for coupling meiotic DSB formation with homolog pairing through the essential participation of an axis component with complexes mediating both processes.
View details for DOI 10.1016/j.devce1.2007.11.016
View details for Web of Science ID 000253241400015
View details for PubMedID 18267094
Finding and keeping your partner during meiosis
2004; 3 (8): 1014-1016
When animals show both frequent innovation and fast social learning, new behaviours can spread more rapidly through populations and potentially increase rates of natural selection and speciation, as proposed by A.C. Wilson in his behavioural drive hypothesis. Comparative work on primates suggests that more innovative species also show more social learning. In this study, we look at intra-specific variation in innovation and social learning in captive wild-caught pigeons. Performances on an innovative problem-solving task and a social learning task are positively correlated in 42 individuals. The correlation remains significant when the effects of neophobia on the two abilities are removed. Neither sex nor dominance rank are associated with performance on the two tasks. Free-flying flocks of urban pigeons are able to solve the innovative food-finding problem used on captive birds, demonstrating it is within the range of their natural capacities. Taken together with the comparative literature, the positive correlation between innovation and social learning suggests that the two abilities are not traded-off.
View details for DOI 10.1007/s10071-006-0064-1
View details for Web of Science ID 000246138500018
View details for PubMedID 17205290
HIM-3 is a meiosis-specific protein that localizes to the cores of chromosomes from the earliest stages of prophase I until the metaphase to anaphase I transition in Caenorhabditis elegans. him-3 mutations disrupt homolog alignment, synapsis, and recombination and we propose that the association of HIM-3 with chromosome axes is a critical event in meiotic chromosome morphogenesis that is required for the proper coordination of these processes. The presence of HIM-3-like proteins in other eukaryotes, some of which are known to be required for synapsis and recombination, suggests the existence of a conserved class of axis-associated proteins that function at the junction of essential meiotic processes.
View details for Web of Science ID 000224088700014
View details for PubMedID 15280669