SUMMARY Decades of progress in developmental cardiology has advanced our understanding of the early aspects of heart development, including cardiomyocyte (CM) differentiation. However, control of CM maturation which is subsequently required to generate adult myocytes, remains elusive. Here, we analyzed over 200 microarray datasets from early embryonic to adult hearts and identified a large number of genes whose expression shifts gradually and continuously during maturation. We generated an atlas of integrated gene expression, biological pathways, transcriptional regulators, and gene regulatory networks (GRNs), which show discrete sets of key transcriptional regulators and pathways activated or suppressed during CM maturation. We developed a GRN-based program named MatStatCM that indexes CM maturation status. MatStatCM reveals that pluripotent stem cell-derived CMs mature early in culture, but are arrested at the late embryonic stage with aberrant regulation of key transcription factors. Our study provides a foundation for understanding CM maturation.
The discovery of the first heart field (FHF) and the second heart field (SHF) led us to understand how cardiac lineages and structures arise during development. However, it remains unknown how they are specified. Here, we generate precardiac spheroids with pluripotent stem cells (PSCs) harboring GFP/RFP reporters under the control of FHF/SHF markers, respectively. GFP+ cells and RFP+ cells appear from two distinct areas and develop in a complementary fashion. Transcriptome analysis shows a high degree of similarities with embryonic FHF/SHF cells. Bmp and Wnt are among the most differentially regulated pathways, and gain- and loss-of-function studies reveal that Bmp specifies GFP+ cells and RFP+ cells via the Bmp/Smad pathway and Wnt signaling, respectively. FHF/SHF cells can be isolated without reporters by the surface protein Cxcr4. This study provides novel insights into understanding the specification of two cardiac origins, which can be leveraged for PSC-based modeling of heart field/chamber-specific disease.
Cardiomyocytes undergo significant structural and functional changes after birth, and these fundamental processes are essential for the heart to pump blood to the growing body. However, due to the challenges of isolating single postnatal/adult myocytes, how individual newborn cardiomyocytes acquire multiple aspects of the mature phenotype remains poorly understood. Here we implement large-particle sorting and analyze single myocytes from neonatal to adult hearts. Early myocytes exhibit wide-ranging transcriptomic and size heterogeneity that is maintained until adulthood with a continuous transcriptomic shift. Gene regulatory network analysis followed by mosaic gene deletion reveals that peroxisome proliferator-activated receptor coactivator-1 signaling, which is active in vivo but inactive in pluripotent stem cell-derived cardiomyocytes, mediates the shift. This signaling simultaneously regulates key aspects of cardiomyocyte maturation through previously unrecognized proteins, including YAP1 and SF3B2. Our study provides a single-cell roadmap of heterogeneous transitions coupled to cellular features and identifies a multifaceted regulator controlling cardiomyocyte maturation.
Myocyte death occurs in many inherited and acquired cardiomyopathies, including arrhythmogenic cardiomyopathy (ACM), a genetic heart disease plagued by the prevalence of sudden cardiac death. Individuals with ACM and harboring pathogenic desmosomal variants, such as desmoglein-2 (DSG2), often show myocyte necrosis with progression to exercise-associated heart failure. Here, we showed that homozygous Dsg2 mutant mice (Dsg2mut/mut), a model of ACM, die prematurely during swimming and display myocardial dysfunction and necrosis. We detected calcium (Ca2+) overload in Dsg2mut/mut hearts, which induced calpain-1 (CAPN1) activation, association of CAPN1 with mitochondria, and CAPN1-induced cleavage of mitochondrial-bound apoptosis-inducing factor (AIF). Cleaved AIF translocated to the myocyte nucleus triggering large-scale DNA fragmentation and cell death, an effect potentiated by mitochondrial-driven AIF oxidation. Posttranslational oxidation of AIF cysteine residues was due, in part, to a depleted mitochondrial thioredoxin-2 redox system. Hearts from exercised Dsg2mut/mut mice were depleted of calpastatin (CAST), an endogenous CAPN1 inhibitor, and overexpressing CAST in myocytes protected against Ca2+ overload–induced necrosis. When cardiomyocytes differentiated from Dsg2mut/mut embryonic stem cells (ES-CMs) were challenged with β-adrenergic stimulation, CAPN1 inhibition attenuated CAPN1-induced AIF truncation. In addition, pretreatment of Dsg2mut/mut ES-CMs with an AIF-mimetic peptide, mirroring the cyclophilin-A (PPIA) binding site of AIF, blocked PPIA-mediated AIF-nuclear translocation, and reduced both apoptosis and necrosis. Thus, preventing CAPN1-induced AIF-truncation or barring binding of AIF to the nuclear chaperone, PPIA, may avert myocyte death and, ultimately, disease progression to heart failure in ACM and likely other forms of cardiomyopathies.
The immaturity of pluripotent stem cell (PSC)-derived tissues has emerged as a universal problem for their biomedical applications. While efforts have been made to generate adult-like cells from PSCs, direct benchmarking of PSC-derived tissues against in vivo development has not been established. Thus, maturation status is often assessed on an ad-hoc basis. Single cell RNA-sequencing (scRNA-seq) offers a promising solution, though cross-study comparison is limited by dataset-specific batch effects. Here, we developed a novel approach to quantify PSC-derived cardiomyocyte (CM) maturation through transcriptomic entropy. Transcriptomic entropy is robust across datasets regardless of differences in isolation protocols, library preparation, and other potential batch effects. With this new model, we analyzed over 45 scRNA-seq datasets and over 52,000 CMs, and established a cross-study, cross-species CM maturation reference. This reference enabled us to directly compare PSC-CMs with the in vivo developmental trajectory and thereby to quantify PSC-CM maturation status. We further found that our entropy-based approach can be used for other cell types, including pancreatic beta cells and hepatocytes. Our study presents a biologically relevant and interpretable metric for quantifying PSC-derived tissue maturation, and is extensible to numerous tissue engineering contexts.
While pluripotent stem cell-derived cardiomyocytes (PSC-CMs) offer tremendous potential for a range of clinical applications, their use has been constrained by the failure to mature these cells to a fully adult-like phenotype. Extensive efforts are currently underway with the goal to mature PSC-CMs. However, comprehensive metrics to benchmark the maturation status and trajectory of PSC-CMs have not been established. Here, we developed a novel approach to quantify CM maturation through single cell transcriptomic entropy. We found that transcriptomic entropy is robust across datasets regardless of differences in isolation protocols, library preparation methods, and other potential batch effects. We analyzed over 40 single cell RNA-sequencing (scRNA-seq) datasets and over 45,000 CMs to establish a cross-study, cross-species reference of CM maturation based on transcriptomic entropy. We subsequently computed the maturation status of PSC-CMs by direct comparison to in vivo development. Our study presents a robust, interpretable, and easy-to-use metric for quantifying CM maturation. cardiomyocyte | transcriptomic | single cell
Rationale: Single cell RNA sequencing (scRNA-seq) has emerged as a powerful tool to profile the transcriptome at single cell resolution, enabling comprehensive analysis of cellular trajectories and heterogeneity during development and disease. However, the use of scRNA-seq remains limited in cardiac pathology owing to technical difficulties associated with the isolation of single adult cardiomyocytes (CMs).Objective: We investigated the capability of large-particle fluorescence-activated cell sorting (LP-FACS) for isolation of viable single adult CMs. Methods and Results:We found that LP-FACS readily outperforms conventional FACS for isolation of struturally competent CMs, including large CMs. Additionally, LP-FACS enables isolation of fluorescent CMs from mosaic models. Importantly, the sorted CMs allow generation of high-quality scRNA-seq libraries. Unlike CMs isolated via previously utilized fluidic or manual methods, LP-FACisolated CMs generate libraries exhibiting normal levels of mitochondrial transcripts. Moreover, LP-FACS isolated CMs remain functionally competent and can be studied for contractile properties. Conclusions:Our study enables high quality dissection of adult CM biology at single-cell resolution..
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