Cardiomyocyte Cell-Cycle Regulation in Neonatal Large Mammals: Single Nucleus RNA-Sequencing Data Analysis via an Artificial-Intelligence–Based Pipeline

Abstract

Adult mammalian cardiomyocytes have very limited capacity to proliferate and repair the myocardial infarction. However, when apical resection (AR) was performed in pig hearts on postnatal day (P) 1 (ARP1) and acute myocardial infarction (MI) was induced on P28 (MIP28), the animals recovered with no evidence of myocardial scarring or decline in contractile performance. Furthermore, the repair process appeared to be driven by cardiomyocyte proliferation, but the regulatory molecules that govern the ARP1-induced enhancement of myocardial recovery remain unclear. Single-nucleus RNA sequencing (snRNA-seq) data collected from fetal pig hearts and the hearts of pigs that underwent ARP1, MIP28, both ARP1 and MI, or neither myocardial injury were evaluated via autoencoder, cluster analysis, sparse learning, and semisupervised learning. Ten clusters of cardiomyocytes (CM1–CM10) were identified across all experimental groups and time points. CM1 was only observed in ARP1 hearts on P28 and was enriched for the expression of T-box transcription factors 5 and 20 (TBX5 and TBX20, respectively), Erb-B2 receptor tyrosine kinase 4 (ERBB4), and G Protein-Coupled Receptor Kinase 5 (GRK5), as well as genes associated with the proliferation and growth of cardiac muscle. CM1 cardiomyocytes also highly expressed genes for glycolysis while lowly expressed genes for adrenergic signaling, which suggested that CM1 were immature cardiomyocytes. Thus, we have identified a cluster of cardiomyocytes, CM1, in neonatal pig hearts that appeared to be generated in response to AR injury on P1 and may have been primed for activation of CM cell-cycle activation and proliferation by the upregulation of TBX5, TBX20, ERBB4, and GRK5.

WINNER: A network biology tool for biomolecular characterization and prioritization

“…Then, prioritization needs to be performed only using the domainknowledge available network to generate hypotheses. Cardiac regeneration, which focuses on cardiomyocyte proliferation, case-study is an example when a significant biological process, not a disease, that does not naturally happen in matured mammals (Porrello et al, 2011;Lam and Sadek, 2018;Ye et al, 2018;Zhu et al, 2018;Zhao et al, 2020;Nakada et al, 2021;Nguyen et al, 2022). In this case, the focus is finding the regulating mechanism to create new cells and to apply this knowledge in biomedical engineering research.…”

Analysis of cardiac single-cell RNA-sequencing data can be improved by the use of artificial-intelligence-based tools

“…AI Autoencoding identified 10 cardiomyocyte clusters (denoted CM1-CM10), one of which (CM1) comprised 62.91% of the cardiomyocytes present in AR P1 hearts on P28 but was essentially absent in all other injury groups and at all other timepoints. In comparison, two other clusters (CM2 and CM10) collectively encompassed 89.62% of cardiomyocytes in AR P1 MI P28 hearts on P30 40 . Notably, CM1 cardiomyocytes were also enriched for the expression of three genes (TBX5 68 , 69 , TBX20 70 , 71 , and ERBB4 72 ) that contribute to the proliferation of cardiomyocytes in fetal and neonatal mouse hearts.…”

“…CM1 cells for which y > 0.1 were categorized as CM1→2, CM1 cells for which y < − 0.1 were categorized as CM1→10, and all other CM1 cells were categorized as inconclusive; then, CM2 cells were combined with CM1→2 cells (CM2 + 1→2), CM10 cells were combined with C1→10 cells (CM10 + 1→10), w and b were re-computed via formulas 2 – 4 with the combined cell populations serving as the predefined positive (CM2 + 1→2, y = 1) and negative (CM10 + 1→10, y = − 1) cell populations, y was recalculated for CM1 cells, and the procedure was repeated until the CM1→2, CM1→10, and inconclusive categories did not change. The results from our AI Semisupervised Learning Model 40 indicated that most (84.78%) CM1 cardiomyocytes would likely follow the CM1→2 trajectory, while the remainder (15.22%) followed the CM1→10 trajectory 40 .…”

“…In the pig scRNAseq experiment, the technical performances were evaluated by reproducing the cardiomyocyte subpopulations that were validated in reference 40 . Briefly, a cardiomyocyte subpopulation, denoted 'CM1' (6537 cells), which was exclusive to the regenerative heart, was found.…”

“…After clustering the pig's cardiomyocytes, similar to 40 , the AI-based sparse support vector technique (sparse model) was applied to quantify cell-cycle phases and proliferative-supporting signaling pathways, including MAPK and HIPPO/YAP signaling. Here, Fetal cardiomyocytes were chosen as 'positive,' and CTL-P56 cardiomyocyte was chosen as 'negative' cells for computing the sparse model.…”