Project description:We have demonstrated previously that adult cardiomyocytes can dedifferentiate and proliferate when cultured in vitro. To determine if cardiomyocyte dedifferentiation and cell cycling/proliferation happens in vivo, we applied here a novel multi-reporter transgenic mouse model (aMH-CMerCreMer;mT/MG;aMHC-H2BBFP) carrying reporter genes for permanent cardiomyocyte lineage mapping and maturity (dedifferentiation) reporting. With this new model, we deciphered the cellular sources and processes of cardiomyocyte dedifferentiation and proliferation in adult hearts. In this study, we used single-nucleus RNA-sequencing to tackle the challenges in analyzing the highly heterogeneous heart cell populations, and obtained datasets for a large number of cardiac single nuclei (both myocytes and non-myocytes) for control and post-infarct hearts. We identified specific cell populations in the heart using distinct transcriptomic clusters, transgenic reporters for ACM lineage and dedifferentiation, as well as cell cycle markers. The results demonstrated that the dedifferentiation and cell cycle progression of pre-existing CMs was augmented in post-infarct hearts, with a number of signaling pathways and gene sets affected. This is the first study dissecting the transcriptomic profiles and signaling pathways associated with cardiomyocyte dedifferentiation and cycling/proliferation in vivo using unbiased high-throughput single-nucleus RNA-Seq analysis, in junction with novel cell lineage (e.g. cardiomyocyte) and phenotyping (e.g. dedifferentiation) transgenic model systems.
Project description: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).
Project description:Rationale: In 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. Objective: To determine the effect of Cfib conditioned media (CM) from the PH calf (PH-CM) on adult rat ventricular myocytes (ARVM) in culture. Methods and Results: Brief 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. Conclusions: These 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:Near-complete reversal of ERBB2-driven cardiomyocyte dedifferentiation is driven by the Hippo pathway, restoring contractility whilst long-lasting conferring cardioprotection.
Project description:In highly regenerative animals, cardiac regeneration occurs innately through cardiomyocyte dedifferentiation and proliferation, although the regenerative mechanisms remain unclear. This project contains processed data sets that were used to identify that klf1, a Kruppel-like transcription factor essential for red blood cell development, is also necessary and sufficient in the myocardium for the induction of cardiomyocyte dedifferentiation and proliferation in adult zebrafish hearts. Raw data has been deposited to PRJNA551130
Project description:The purpose of this study was to understand differences between maturation processes in endogenous and pluripotent stem cell-derived cardiomyocytes. To this end, we first expanded our previous reference of endogenous cardiomyocyte maturation, isolated by LP-FACS (see Kannan et al., bioRxiv 2020, and GSE147807). We subsequently generated PSCs from the same background strain and differentiated to CMs. CMs were isolated by conventional FACs.
Project description:Cardiac metabolism plays a crucial role in producing sufficient energy to sustain cardiac function. However, the role of metabolism in different aspects of cardiomyocyte regeneration remains unclear. Working with the adult zebrafish heart regeneration model, we first find an increase in the levels of mRNAs encoding enzymes regulating glucose and pyruvate metabolism, including pyruvate kinase M1/2 (Pkm) and pyruvate dehydrogenase kinases (Pdks), especially in tissues bordering the damaged area. We further find that impaired glycolysis decreases the number of proliferating cardiomyocytes following injury. These observations are supported by analyses using loss-of-function models for the metabolic regulators Pkma2 and peroxisome proliferator-activated receptor gamma coactivator 1 alpha. Cardiomyocyte-specific loss- and gain-of-function manipulations of pyruvate metabolism using Pdk3 as well as a catalytic subunit of the pyruvate dehydrogenase complex (PDC) reveal its importance in cardiomyocyte dedifferentiation and proliferation after injury. Furthermore, we find that PDK activity can modulate cell cycle progression and protrusive activity in mammalian cardiomyocytes in culture. Our findings reveal new roles for cardiac metabolism and the PDK-PDC axis in cardiomyocyte behavior following cardiac injury.