Cardiac Development at a Single-Cell Resolution.

Mammalian cardiac development is a complex, multistage process. Though traditional lineage tracing studies have characterized the broad trajectories of cardiac progenitors, the advent and rapid optimization of single-cell RNA sequencing methods have yielded an ever-expanding toolkit for characterizing heterogeneous cell populations in the developing heart. Importantly, they have allowed for a robust profiling of the spatiotemporal transcriptomic landscape of the human and mouse heart, revealing the diversity of cardiac cells-myocyte and non-myocyte-over the course of development. These studies have yielded insights into novel cardiac progenitor populations, chamber-specific developmental signatures, the gene regulatory networks governing cardiac development, and, thus, the etiologies of congenital heart diseases. Furthermore, single-cell RNA sequencing has allowed for the exquisite characterization of distinct cardiac populations such as the hard-to-capture cardiac conduction system and the intracardiac immune population. Therefore, single-cell profiling has also resulted in new insights into the regulation of cardiac regeneration and injury repair. Single-cell multiomics approaches combining transcriptomics, genomics, and epigenomics may uncover an even more comprehensive atlas of human cardiac biology. Single-cell analyses of the developing and adult mammalian heart offer an unprecedented look into the fundamental mechanisms of cardiac development and the complex diseases that may arise from it.

The cardiac conduction system: History, development, and disease.

The heart is the first organ to form during embryonic development, establishing the circulatory infrastructure necessary to sustain life and enable downstream organogenesis. Critical to the heart's function is its ability to initiate and propagate electrical impulses that allow for the coordinated contraction and relaxation of its chambers, and thus, the movement of blood and nutrients. Several specialized structures within the heart, collectively known as the cardiac conduction system (CCS), are responsible for this phenomenon. In this review, we discuss the discovery and scientific history of the mammalian cardiac conduction system as well as the key genes and transcription factors implicated in the formation of its major structures. We also describe known human diseases related to CCS development and explore existing challenges in the clinical context.

Multicenter Study of Survival Benefit of Cardiac Resynchronization Therapy in Pediatric and Congenital Heart Disease.

BACKGROUND: Evidence for the efficacy of cardiac resynchronization therapy (CRT) in pediatric and congenital heart disease (CHD) has been limited to surrogate outcomes. OBJECTIVES: This study aimed to assess the impact of CRT upon the risk of transplantation or death in a retrospective, high-risk, controlled cohort at 5 quaternary referral centers. METHODS: Both CRT patients and control patients were <21 years of age or had CHD; had systemic ventricular ejection fraction <45%; symptomatic heart failure; and significant electrical dyssynchrony (QRS duration z score >3 or single-site ventricular pacing >40%) at enrollment. Patients with CRT were matched with control patients via 1:1 propensity score matching. CRT patients were enrolled at CRT implantation; control patients were enrolled at the outpatient clinical encounter where inclusion criteria were first met. The primary endpoint was transplantation or death. RESULTS: In total, 324 control patients and 167 CRT recipients were identified. Mean follow-up was 4.2 ± 3.7 years. Upon propensity score matching, 139 closely matched pairs were identified (20 baseline indices). Of the 139 matched pairs, 52 (37.0%) control patients and 31 (22.0%) CRT recipients reached the primary endpoint. On both unadjusted and multivariable Cox regression analysis, the risk reduction associated with CRT for the primary endpoint was significant (HR: 0.40; 95% CI: 0.25-0.64; P < 0.001; and HR: 0.44; 95% CI: 0.28-0.71; P = 0.001, respectively). On longitudinal assessment, the CRT group had significantly improved systemic ventricular ejection fraction (P < 0.001) and shorter QRS duration (P = 0.015), sustained to 5 years. CONCLUSIONS: In pediatric and CHD patients with symptomatic systolic heart failure and electrical dyssynchrony, CRT was associated with improved heart transplantation-free survival.

Utility of smart watches for identifying arrhythmias in children.

BACKGROUND: Arrhythmia symptoms are frequent complaints in children and often require a pediatric cardiology evaluation. Data regarding the clinical utility of wearable technologies are limited in children. We hypothesize that an Apple Watch can capture arrhythmias in children. METHODS: We present an analysis of patients ≤18 years-of-age who had signs of an arrhythmia documented by an Apple Watch. We include patients evaluated at our center over a 4-year-period and highlight those receiving a formal arrhythmia diagnosis. We evaluate the role of the Apple Watch in arrhythmia diagnosis, the results of other ambulatory cardiac monitoring studies, and findings of any EP studies. RESULTS: We identify 145 electronic-medical-record identifications of Apple Watch, and find arrhythmias confirmed in 41 patients (28%) [mean age 13.8 ± 3.2 years]. The arrythmias include: 36 SVT (88%), 3 VT (7%), 1 heart block (2.5%) and wide 1 complex tachycardia (2.5%). We show that invasive EP study confirmed diagnosis in 34 of the 36 patients (94%) with SVT (2 non-inducible). We find that the Apple Watch helped prompt a workup resulting in a new arrhythmia diagnosis for 29 patients (71%). We note traditional ambulatory cardiac monitors were worn by 35 patients (85%), which did not detect arrhythmias in 10 patients (29%). In 73 patients who used an Apple Watch for recreational or self-directed heart rate monitoring, 18 (25%) sought care due to device findings without any arrhythmias identified. CONCLUSION: We demonstrate that the Apple Watch can record arrhythmia events in children, including events not identified on traditionally used ambulatory monitors.

Wearable devices, such as smart watches, have become popular for the monitoring of health, particularly for people with heart conditions. Wearable devices have been well-studied in adults, however there is less information available on their effectiveness in monitoring children's health. We reviewed the heart electrical recordings of a group of children who submitted recordings obtained from their Apple Watches during moments when they felt as though their heart's rhythm was abnormal. The Apple Watches captured rhythm abnormalities that matched the diagnoses obtained using heart monitors used clinically. This study shows that use of Apple Watches can enable clinicians to identify abnormalities that many traditional at-home monitoring devices do not detect. Thus, wearable devices, such as the Apple Watch, could be used to help identify heart rhythm disorders in children.

Combined lineage tracing and scRNA-seq reveals unexpected first heart field predominance of human iPSC differentiation.

During mammalian development, the left and right ventricles arise from early populations of cardiac progenitors known as the first and second heart fields, respectively. While these populations have been extensively studied in non-human model systems, their identification and study in vivo human tissues have been limited due to the ethical and technical limitations of accessing gastrulation-stage human embryos. Human-induced pluripotent stem cells (hiPSCs) present an exciting alternative for modeling early human embryogenesis due to their well-established ability to differentiate into all embryonic germ layers. Here, we describe the development of a TBX5/MYL2 lineage tracing reporter system that allows for the identification of FHF- progenitors and their descendants including left ventricular cardiomyocytes. Furthermore, using single-cell RNA sequencing (scRNA-seq) with oligonucleotide-based sample multiplexing, we extensively profiled differentiating hiPSCs across 12 timepoints in two independent iPSC lines. Surprisingly, our reporter system and scRNA-seq analysis revealed a predominance of FHF differentiation using the small molecule Wnt-based 2D differentiation protocol. We compared this data with existing murine and 3D cardiac organoid scRNA-seq data and confirmed the dominance of left ventricular cardiomyocytes (>90%) in our hiPSC-derived progeny. Together, our work provides the scientific community with a powerful new genetic lineage tracing approach as well as a single-cell transcriptomic atlas of hiPSCs undergoing cardiac differentiation.

Outcomes After Development of Ventricular Arrhythmias in Single Ventricular Heart Disease Patients With Fontan Palliation.

BACKGROUND: With the advent of more intensive rhythm monitoring strategies, ventricular arrhythmias (VAs) are increasingly detected in Fontan patients. However, the prognostic implications of VA are poorly understood. We assessed the incidence of VA in Fontan patients and the implications on transplant-free survival. METHODS: Medical records of Fontan patients seen at a single center between 2002 and 2019 were reviewed to identify post-Fontan VA (nonsustained ventricular tachycardia >4 beats or sustained >30 seconds). Patients with preFontan VA were excluded. Hemodynamically unstable VA was defined as malignant VA. The primary outcome was death and heart transplantation. Death with censoring at transplant was a secondary outcome. RESULTS: Of 431 Fontan patients, transplant-free survival was 82% at 15 years post-Fontan with 64 (15%) meeting primary outcome of either death (n=16, 3.7%), at a median 4.6 (0.4-10.2) years post-Fontan, or transplant (n=48, 11%), at a median of 11.1 (5.9-16.2) years post-Fontan. Forty-eight (11%) patients were diagnosed with VA (90% nonsustained ventricular tachycardia, 10% sustained ventricular tachycardia). Malignant VA (n=9, 2.0%) was associated with younger age, worse systolic function, and valvular regurgitation. Risk for VA increased with time from Fontan, 2.4% at 10 years to 19% at 20 years. History of Stage 1 surgery with right ventricular to pulmonary artery conduit and older age at Fontan were significant risk factors for VA. VA was strongly associated with an increased risk of transplant or death (HR, 9.2 [95% CI, 4.5-18.7]; P<0.001), with a transplant-free survival of 48% at 5-year post-VA diagnosis. CONCLUSIONS: Ventricular arrhythmias occurred in 11% of Fontan patients and was highly associated with transplant or death, with a transplant-free survival of <50% at 5-year post-VA diagnosis. Risk factors for VA included older age at Fontan and history of right ventricular to pulmonary artery conduit. A diagnosis of VA in Fontan patients should prompt increased clinical surveillance.

devCellPy is a machine learning-enabled pipeline for automated annotation of complex multilayered single-cell transcriptomic data.

A major informatic challenge in single cell RNA-sequencing analysis is the precise annotation of datasets where cells exhibit complex multilayered identities or transitory states. Here, we present devCellPy a highly accurate and precise machine learning-enabled tool that enables automated prediction of cell types across complex annotation hierarchies. To demonstrate the power of devCellPy, we construct a murine cardiac developmental atlas from published datasets encompassing 104,199 cells from E6.5-E16.5 and train devCellPy to generate a cardiac prediction algorithm. Using this algorithm, we observe a high prediction accuracy (>90%) across multiple layers of annotation and across de novo murine developmental data. Furthermore, we conduct a cross-species prediction of cardiomyocyte subtypes from in vitro-derived human induced pluripotent stem cells and unexpectedly uncover a predominance of left ventricular (LV) identity that we confirmed by an LV-specific TBX5 lineage tracing system. Together, our results show devCellPy to be a useful tool for automated cell prediction across complex cellular hierarchies, species, and experimental systems.

In vivo visualization and molecular targeting of the cardiac conduction system.

Accidental injury to the cardiac conduction system (CCS), a network of specialized cells embedded within the heart and indistinguishable from the surrounding heart muscle tissue, is a major complication in cardiac surgeries. Here, we addressed this unmet need by engineering targeted antibody-dye conjugates directed against the CCS, allowing for the visualization of the CCS in vivo following a single intravenous injection in mice. These optical imaging tools showed high sensitivity, specificity, and resolution, with no adverse effects on CCS function. Further, with the goal of creating a viable prototype for human use, we generated a fully human monoclonal Fab that similarly targets the CCS with high specificity. We demonstrate that, when conjugated to an alternative cargo, this Fab can also be used to modulate CCS biology in vivo, providing a proof of principle for targeted cardiac therapeutics. Finally, in performing differential gene expression analyses of the entire murine CCS at single-cell resolution, we uncovered and validated a suite of additional cell surface markers that can be used to molecularly target the distinct subcomponents of the CCS, each prone to distinct life-threatening arrhythmias. These findings lay the foundation for translational approaches targeting the CCS for visualization and therapy in cardiothoracic surgery, cardiac imaging, and arrhythmia management.

The Tabula Sapiens: A multiple-organ, single-cell transcriptomic atlas of humans.

Molecular characterization of cell types using single-cell transcriptome sequencing is revolutionizing cell biology and enabling new insights into the physiology of human organs. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues, and tissue-specific variation in gene expression. Using multiple tissues from a single donor enabled identification of the clonal distribution of T cells between tissues, identification of the tissue-specific mutation rate in B cells, and analysis of the cell cycle state and proliferative potential of shared cell types across tissues. Cell type-specific RNA splicing was discovered and analyzed across tissues within an individual.

Implantable Cardioverter Defibrillators in Infants and Toddlers: Indications, Placement, Programming, and Outcomes.

BACKGROUND: Limited data exist regarding implantable cardioverter defibrillator (ICD) usage in infants and toddlers. This study evaluates ICD placement indications, procedural techniques, programming strategies, and outcomes of ICDs in infants and toddlers. METHODS: This is a single-center retrospective review of all patients ≤3 years old who received an ICD from 2009 to 2021. RESULTS: Fifteen patients received an ICD at an age of 1.2 years (interquartile range [IQR], 0.1-2.4; 12 [80%] women; weight, 8.2 kg [IQR, 4.2-12.6]) and were followed for a median of 4.28 years (IQR, 1.40-5.53) or 64.2 patient-years. ICDs were placed for secondary prevention in 12 patients (80%). Diagnoses included 8 long-QT syndromes (53%), 4 idiopathic ventricular tachycardias/ventricular fibrillations (VFs; 27%), 1 recurrent ventricular tachycardia with cardiomyopathy (7%), 1 VF with left ventricular noncompaction (7%), and 1 catecholaminergic polymorphic ventricular tachycardia (7%). All implants were epicardial, with a coil in the pericardial space. Intraoperative defibrillation safety testing was attempted in 11 patients (73%), with VF induced in 8 (53%). Successful restoration of sinus rhythm was achieved in all tested patients with a median of 9 (IQR, 7.3-11.3) J or 0.90 (IQR, 0.68-1.04) J/kg. Complications consisted of 1 postoperative chylothorax and 3 episodes of feeding intolerance. VF detection was programmed to 250 (IQR, 240-250) ms with first shock delivering 10 (IQR, 5-15) J or 1.1 (IQR, 0.8-1.4) J/kg. Three patients (20%) received appropriate shocks for ventricular tachycardia/VF. No patient received an inappropriate shock. There were 2 (13%) ventricular lead fractures (at 2.6 and 4.2 years post-implant), 1 (7%) pocket-site infection, and 2 (13%) generator exchanges. All patients were alive, and 1 patient (7%) received a heart transplant. CONCLUSIONS: ICDs can be safely and effectively placed for sudden death prevention in infants and toddlers with good midterm outcomes.

Identifying an appropriate endpoint for cryoablation in children with atrioventricular nodal reentrant tachycardia: Is residual slow pathway conduction associated with recurrence?

BACKGROUND: Cryoablation is increasingly used to treat atrioventricular nodal reentrant tachycardia (AVNRT) due to its safety profile. However, cryoablation may have higher recurrence than radiofrequency ablation (RFA), and the optimal procedural endpoint remains undefined. OBJECTIVE: The purpose of this study was to identify the association of cryoablation procedural endpoints with postprocedural AVNRT recurrence. METHODS: We performed a single-center, retrospective analysis of pediatric patients following successful first-time cryoablation for AVNRT between January 1, 2011, and December 31, 2019. Preablation inducibility of AVNRT was recorded. Procedural endpoints, including slow pathway (SP) conduction (presence of jump or echo beats) with and without isoproterenol, were identified. Recurrence was established from clinical notes and/or direct patient contact. RESULTS: Of 256 patients, 147 (57%) were assessed on isoproterenol precryoablation, and 171 (47%) were assessed on isoproterenol postcryoablation. Mean cryolesion time was 2586 ± 1434 seconds. Following ablation, 104 (41%) had some evidence of residual SP conduction. With median follow-up time of 1.9 [0.7-3.7] years, recurrence occurred in 14 patients (5%). Complete elimination of SP conduction (with and without isoproterenol) had a hazard ratio for recurrence of 1.26 (95% confidence interval [CI] 0.42-3.8; P = .68) on univariate analysis and 1.39 (95% CI 0.36-5.4; P = .63) on multivariate analysis (including demographics, ablation time, 8-mm cryocatheter, and baseline inducibility). CONCLUSION: The observed AVNRT recurrence rate after cryoablation was comparable to that of RFA. The presence of residual SP conduction was not associated with recurrence. This suggests that jump or single echo beat may be an acceptable endpoint in AVNRT cryoablation.

Molecular Profiling of the Cardiac Conduction System: the Dawn of a New Era.

PURPOSE OF REVIEW: Recent technological advances have led to an increased ability to define the gene expression profile of the cardiac conduction system (CCS). Here, we review the most salient studies to emerge in recent years and discuss existing gaps in our knowledge as well as future areas of investigation. RECENT FINDINGS: Molecular profiling of the CCS spans several decades. However, the advent of high-throughput sequencing strategies has allowed for the discovery of unique transcriptional programs of the many diverse CCS cell types. The CCS, a diverse structure with significant inter- and intra-component cellular heterogeneity, is essential to the normal function of the heart. Progress in transcriptomic profiling has improved the resolution and depth of characterization of these unique and clinically relevant CCS cell types. Future studies leveraging this big data will play a crucial role in improving our understanding of CCS development and function as well as translating these findings into tangible translational tools for the improved detection, prevention, and treatment of cardiac arrhythmias.

CRISPR/Cas9-based targeting of fluorescent reporters to human iPSCs to isolate atrial and ventricular-specific cardiomyocytes.

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.

Massive expansion and cryopreservation of functional human induced pluripotent stem cell-derived cardiomyocytes.

Since the discovery of human induced pluripotent stem cells (hiPSCs), numerous strategies have been established to efficiently derive cardiomyocytes from hiPSCs (hiPSC-CMs). Here, we describe a cost-effective strategy for the subsequent massive expansion (>250-fold) of high-purity hiPSC-CMs relying on two aspects: removal of cell-cell contacts and small-molecule inhibition with CHIR99021. The protocol maintains CM functionality, allows cryopreservation, and the cells can be used in downstream assays such as disease modeling, drug and toxicity screening, and cell therapy. For complete details on the use and execution of this protocol, please refer to Buikema (2020).

Wnt Activation and Reduced Cell-Cell Contact Synergistically Induce Massive Expansion of Functional Human iPSC-Derived Cardiomyocytes.

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-3ß (GSK-3ß) inhibition using CHIR99021 and concurrent removal of cell-cell contact. We show that GSK-3ß 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-3ß 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.

Pectoral nerve blocks decrease postoperative pain and opioid use after pacemaker or implantable cardioverter-defibrillator placement in children.

BACKGROUND: Pectoral nerve blocks (PECs) can reduce intraprocedural anesthetic requirements and postoperative pain. Little is known about the utility of PECs in reducing pain and narcotic use after pacemaker (PM) or implantable cardioverter-defibrillator (ICD) placement in children. OBJECTIVE: The purpose of this study was 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 and 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 had undergone PECs and those who had undergone conventional local anesthetic (Control). RESULTS: A total of 74 patients underwent PM or ICD placement; 20 patients (27%) underwent 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% confidence interval [CI] 0.8-2.2) vs Control 3.1 (95% CI 2.7-3.5); P <.001] and lower total opioid use [PECs 6.0 morphine milligram equivalent (MME)/m2 (95% CI 3.4-8.6) vs Control 15.0 MME/m2 (95% CI 11.8-18.2); P = .001] over the 24 hours postimplant. CONCLUSION: PECs 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.

Transcriptomic Profiling of the Developing Cardiac Conduction System at Single-Cell Resolution.

RATIONALE: The cardiac conduction system (CCS) consists of distinct components including the sinoatrial node, atrioventricular node, His bundle, bundle branches, and Purkinje fibers. Despite an essential role for the CCS in heart development and function, the CCS has remained challenging to interrogate because of inherent obstacles including small cell numbers, large cell-type heterogeneity, complex anatomy, and difficulty in isolation. Single-cell RNA-sequencing 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 single-cell RNA-sequencing within the developing mouse heart. METHODS AND RESULTS: Wild-type, embryonic day 16.5 mouse hearts (n=6 per zone) were harvested and 3 zones of microdissection were isolated, including: Zone I-sinoatrial node region; Zone II-atrioventricular node/His region; and Zone III-bundle branch/Purkinje fiber region. Tissue was digested into single-cell suspensions, cells isolated, mRNA reverse transcribed, and barcoded before high-throughput sequencing and bioinformatics analyses. Single-cell RNA-sequencing 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 coexpression 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 immunolabeling with volume imaging (iDISCO+) in 3 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 characterization in the context of development and disease.

Single cell expression analysis reveals anatomical and cell cycle-dependent transcriptional shifts during heart development.

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 have 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.