Dataset Information

Single-cell imaging and transcriptomic analyses of endogenous cardiomyocyte dedifferentiation and cycling.

ABSTRACT: While it is recognized that there are low levels of new cardiomyocyte (CM) formation throughout life, the source of these new CM generates much debate. One hypothesis is that these new CMs arise from the proliferation of existing CMs potentially after dedifferentiation although direct evidence for this is lacking. Here we explore the mechanisms responsible for CM renewal in vivo using multi-reporter transgenic mouse models featuring efficient adult CM (ACM) genetic cell fate mapping and real-time cardiomyocyte lineage and dedifferentiation reporting. Our results demonstrate that non-myocytes (e.g., cardiac progenitor cells) contribute negligibly to new ACM formation at baseline or after cardiac injury. In contrast, we found a significant increase in dedifferentiated, cycling CMs in post-infarct hearts. ACM cell cycling was enhanced within the dedifferentiated CM population. Single-nucleus transcriptomic analysis demonstrated that CMs identified with dedifferentiation reporters had significant down-regulation in gene networks for cardiac hypertrophy, contractile, and electrical function, with shifts in metabolic pathways, but up-regulation in signaling pathways and gene sets for active cell cycle, proliferation, and cell survival. The results demonstrate that dedifferentiation may be an important prerequisite for CM proliferation and explain the limited but measurable cardiac myogenesis seen after myocardial infarction (MI).

SUBMITTER: Zhang Y

PROVIDER: S-EPMC6547664 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Single-cell imaging and transcriptomic analyses of endogenous cardiomyocyte dedifferentiation and cycling.

Zhang Yiqiang Y Gago-Lopez Nuria N Li Ning N Zhang Zhenhe Z Alver Naima N Liu Yonggang Y Martinson Amy M AM Mehri Avin A MacLellan William Robb WR

Cell discovery 20190604

While it is recognized that there are low levels of new cardiomyocyte (CM) formation throughout life, the source of these new CM generates much debate. One hypothesis is that these new CMs arise from the proliferation of existing CMs potentially after dedifferentiation although direct evidence for this is lacking. Here we explore the mechanisms responsible for CM renewal in vivo using multi-reporter transgenic mouse models featuring efficient adult CM (ACM) genetic cell fate mapping and real-tim ...[more]

PMID: 31231540

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Project description:RationaleIn virtually all models of heart failure, prognosis is determined by right ventricular (RV) function; thus, understanding the cellular mechanisms contributing to RV dysfunction is critical. Whole organ remodeling is associated with cell-specific changes, including cardiomyocyte dedifferentiation and activation of cardiac fibroblasts (Cfib) which in turn is linked to disorganization of cytoskeletal proteins and loss of sarcomeric structures. However, how these cellular changes contribute to RV function remains unknown. We've previously shown significant organ-level RV dysfunction in a large animal model of pulmonary hypertension (PH) which was not mirrored by reduced function of isolated cardiomyocytes. We hypothesized that factors produced by the endogenous Cfib contribute to global RV dysfunction by generating a heterogeneous cellular environment populated by dedifferentiated cells.ObjectiveTo determine the effect of Cfib conditioned media (CM) from the PH calf (PH-CM) on adult rat ventricular myocytes (ARVM) in culture.Methods and resultsBrief exposure (<2 days) to PH-CM results in rapid, marked dedifferentiation of ARVM to a neonatal-like phenotype exhibiting spontaneous contractile behavior. Dedifferentiated cells maintain viability for over 30 days with continued expression of cardiomyocyte proteins including TnI and α-actinin yet exhibit myofibroblast characteristics including expression of α-smooth muscle actin. Using a bioinformatics approach to identify factor(s) that contribute to dedifferentiation, we found activation of the PH Cfib results in a unique transcriptome correlating with factors both in the secretome and with activated pathways in the dedifferentiated myocyte. Further, we identified upregulation of periostin in the Cfib and CM, and demonstrate that periostin is sufficient to drive cardiomyocyte dedifferentiation.ConclusionsThese data suggest that paracrine factor(s) released by Cfib from the PH calf signal a phenotypic transformation in a population of cardiomyocytes that likely contributes to RV dysfunction. Therapies targeting this process, such as inhibition of periostin, have the potential to prevent RV dysfunction.

Project description:BackgroundRodent cardiomyocytes (CM) undergo mitotic arrest and decline of mononucleated-diploid population post-birth, which are implicated in neonatal loss of heart regenerative potential. However, the dynamics of postnatal CM maturation are largely unknown in swine, despite a similar neonatal cardiac regenerative capacity as rodents. Here, we provide a comprehensive analysis of postnatal cardiac maturation in swine, including CM cell cycling, multinucleation and hypertrophic growth, as well as non-CM cardiac factors such as extracellular matrix (ECM), immune cells, capillaries, and neurons. Our study reveals discordance in postnatal pig heart maturational events compared to rodents.Methods and resultsLeft-ventricular myocardium from White Yorkshire-Landrace pigs at postnatal day (P)0 to 6 months (6mo) was analyzed. Mature cardiac sarcomeric characteristics, such as fetal TNNI1 repression and Cx43 co-localization to cell junctions, were not evident until P30 in pigs. In CMs, appreciable binucleation is observed by P7, with extensive multinucleation (4-16 nuclei per CM) beyond P15. Individual CM nuclei remain predominantly diploid at all ages. CM mononucleation at ~50% incidence is observed at P7-P15, and CM mitotic activity is measurable up to 2mo. CM cross-sectional area does not increase until 2mo-6mo in pigs, though longitudinal CM growth proportional to multinucleation occurs after P15. RNAseq analysis of neonatal pig left ventricles showed increased expression of ECM maturation, immune signaling, neuronal remodeling, and reactive oxygen species response genes, highlighting significance of the non-CM milieu in postnatal mammalian heart maturation.ConclusionsCM maturational events such as decline of mononucleation and cell cycle arrest occur over a 2-month postnatal period in pigs, despite reported loss of heart regenerative potential by P3. Moreover, CMs grow primarily by multinucleation and longitudinal hypertrophy in older pig CMs, distinct from mice and humans. These differences are important to consider for preclinical testing of cardiovascular therapies using swine, and may offer opportunities to study aspects of heart regeneration unavailable in other models.

Project description:Separation of B cells into different subsets has been useful to understand their different functions in various immune scenarios. In some instances, the subsets defined by phenotypic FACS separation are relatively homogeneous and so establishing the functions associated with them is straightforward. Other subsets, such as the "Double negative" (DN, CD19+CD27-IgD-) population, are more complex with reports of differing functionality which could indicate a heterogeneous population. Recent advances in single-cell techniques enable an alternative route to characterize cells based on their transcriptome. To maximize immunological insight, we need to match prior data from phenotype-based studies with the finer granularity of the single-cell transcriptomic signatures. We also need to be able to define meaningful B cell subsets from single cell analyses performed on PBMCs, where the relative paucity of a B cell signature means that defining B cell subsets within the whole is challenging. Here we provide a reference single-cell dataset based on phenotypically sorted B cells and an unbiased procedure to better classify functional B cell subsets in the peripheral blood, particularly useful in establishing a baseline cellular landscape and in extracting significant changes with respect to this baseline from single-cell datasets. We find 10 different clusters of B cells and applied a novel, geometry-inspired, method to RNA velocity estimates in order to evaluate the dynamic transitions between B cell clusters. This indicated the presence of two main developmental branches of memory B cells. A T-independent branch that involves IgM memory cells and two DN subpopulations, culminating in a population thought to be associated with Age related B cells and the extrafollicular response. The other, T-dependent, branch involves a third DN cluster which appears to be a precursor of classical memory cells. In addition, we identify a novel DN4 population, which is IgE rich and closely linked to the classical/precursor memory branch suggesting an IgE specific T-dependent cell population.

Dataset Information

Single-cell imaging and transcriptomic analyses of endogenous cardiomyocyte dedifferentiation and cycling.

Publications

Single-cell imaging and transcriptomic analyses of endogenous cardiomyocyte dedifferentiation and cycling.

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