FUCCI-Based Live Imaging Platform Reveals Cell Cycle Dynamics and Identifies Pro-proliferative Compounds in Human iPSC-Derived Cardiomyocytes.

One of the major goals in cardiac regeneration research is to replace lost ventricular tissue with new cardiomyocytes. However, cardiomyocyte proliferation drops to low levels in neonatal hearts and is no longer efficient in compensating for the loss of functional myocardium in heart disease. We generated a human induced pluripotent stem cell (iPSC)-derived cardiomyocyte-specific cell cycle indicator system (TNNT2-FUCCI) to characterize regular and aberrant cardiomyocyte cycle dynamics. We visualized cell cycle progression in TNNT2-FUCCI and found G2 cycle arrest in endoreplicating cardiomyocytes. Moreover, we devised a live-cell compound screening platform to identify pro-proliferative drug candidates. We found that the alpha-adrenergic receptor agonist clonidine induced cardiomyocyte proliferation in vitro and increased cardiomyocyte cell cycle entry in neonatal mice. In conclusion, the TNNT2-FUCCI system is a versatile tool to characterize cardiomyocyte cell cycle dynamics and identify pro-proliferative candidates with regenerative potential in the mammalian heart.

Microenvironment Stiffness Amplifies Post-ischemia Heart Regeneration in Response to Exogenous Extracellular Matrix Proteins in Neonatal Mice.

The cardiogenesis of the fetal heart is absent in juveniles and adults. Cross-transplantation of decellularized extracellular matrix (dECM) can stimulate regeneration in myocardial infarct (MI) models. We have previously shown that dECM and tissue stiffness have cooperative regulation of heart regeneration in transiently regenerative day 1 neonatal mice. To investigate underlying mechanisms of mechano-signaling and dECM, we pharmacologically altered heart stiffness and administered dECM hydrogels in non-regenerative mice after MI. The dECM combined with softening exhibits preserved cardiac function, LV geometry, increased cardiomyocyte mitosis and lowered fibrosis while stiffening further aggravated ischemic damage. Transcriptome analysis identified a protein in cardiomyocytes, CLCA2, confirmed to be upregulated after MI and downregulated by dECM in a mechanosensitive manner. Synthetic knock-down of CLCA2 expression induced mitosis in primary rat cardiomyocytes in the dish. Together, our results indicate that therapeutic efficacy of extracellular molecules for heart regeneration can be modulated by heart microenvironment stiffness in vivo.

Mechanisms Underlying Cardiomyocyte Development: Can We Exploit Them to Regenerate the Heart?

PURPOSE OF REVIEW: It is well established that the adult mammalian cardiomyocytes retain a low capacity for cell cycle activity; however, it is insufficient to effectively respond to myocardial injury and facilitate cardiac regenerative repair. Lessons learned from species in which cardiomyocytes do allow for proliferative regeneration/repair have shed light into the mechanisms underlying cardiac regeneration post-injury. Importantly, many of these mechanisms are conserved across species, including mammals, and efforts to tap into these mechanisms effectively within the adult heart are currently of great interest. RECENT FINDINGS: Targeting the endogenous gene regulatory networks (GRNs) shown to play roles in the cardiac regeneration of conducive species is seen as a strong approach, as delivery of a single or combination of genes has promise to effectively enhance cell cycle activity and CM proliferation in adult hearts post-myocardial infarction (MI). In situ re-induction of proliferative gene regulatory programs within existing, local, non-damaged cardiomyocytes helps overcome significant technical hurdles, such as successful engraftment of implanted cells or achieving complete cardiomyocyte differentiation from cell-based approaches. Although many obstacles currently exist and need to be overcome to successfully translate these approaches to clinical settings, the current efforts presented here show great promise.

Natriuretic peptides and Forkhead O transcription factors act in a cooperative manner to promote cardiomyocyte cell cycle re-entry in the postnatal mouse heart.

BACKGROUND: Cardiomyocytes proliferate rapidly during fetal life but lose their ability of proliferation soon after birth. However, before terminal withdrawal from the cell cycle, cardiomyocytes undergo another round of cell cycle during early postnatal life in mice. While a transient wave of increased DNA synthesis in cardiomyocyte has been observed in postnatal mouse hearts, the molecular mechanisms describing cardiomyocyte cell cycle re-entry remain poorly understood. Atrial and B-type natriuretic peptides (ANP and BNP) are abundantly expressed in embryonic heart ventricles. After birth, the expression of both genes is strongly reduced in the ventricular myocardium. Forkhead O (FOXO) transcription factors are expressed in both embryonic and postnatal heart ventricles. Their transcriptional activity negatively affects cardiomyocyte proliferation. Upon phosphorylation, FOXO is translocated to the cytoplasm and is transcriptionally inactive. Despite these important findings, it remains largely unknown whether natriuretic peptides and FOXO cooperatively play a role in regulating cardiomyocyte cell cycle activity during early postnatal life. RESULTS: We observed that the expression of ANP and BNP and the level of phosphorylated FOXO were transiently increased in the postnatal mouse heart ventricles, which coincided with the burst of cardiomyocyte cell cycle re-entry during early postnatal life in mice. Cell culture studies showed that ANP/BNP signaling and FOXO cooperatively promoted cell cycle activity in neonatal mouse cardiomyocytes. The enhanced cell cycle activity observed in combined treatment of ANP/BNP and dominant-negative FOXO (DN-FOXO), which can bind FOXO recognition sites on DNA but cannot activate transcription, was primarily mediated through natriuretic peptide receptor 3 (Npr3). In mice, simultaneous application of ANP and DN-FOXO in postnatal hearts reactivated cell cycle in cardiomyocytes, resulting in reduced scar formation after experimental myocardial infarction. CONCLUSIONS: Our data demonstrate the cooperative effects of natriuretic peptide and DN-FOXO on promoting cardiomyocyte cell cycle activity and mouse cardiac repair and regeneration after injury.

High-Fidelity Quantification of Cell Cycle Activity with Multi-Isotope Imaging Mass Spectrometry.

The quantification of cell cycle activity is a prerequisite to defining the dynamics and scope of organ development or regeneration. Multi-isotope imaging mass spectrometry (MIMS) merges stable isotope tracers with an imaging mass spectrometry platform called NanoSIMS, which can quantitatively measure the incorporation of stable isotope tracers with high precision in suborganelle domains. MIMS has been applied to quantify the dynamics of postnatal cardiogenesis and mammalian cardiomyocyte regeneration during aging or in response to injury. Here, we present an approach to the conduct of MIMS experiments, with an emphasis on the application to the field of cardiac regeneration; however, the approach is also applicable, with, at most, minor modifications to broader biological questions.

Low Temperature Affects Stem Cell Maintenance in Brassica oleracea Seedlings.

Most of the above ground tissues in higher plants originate from stem cells located in the shoot apical meristem (SAM). Several plant species can suffer from spontaneous stem cell arrest resulting in lack of further shoot development. In Brassica oleracea this SAM arrest is known as blindness and occurs in an unpredictable manner leading to considerable economic losses for plant raisers and farmers. Detailed analyses of seedlings showed that stem cell arrest is triggered by low temperatures during germination. To induce this arrest reproducibly and to study the effect of the environment, an assay was developed. The role of genetic variation on the susceptibility to develop blind seedlings was analyzed by a quantitative genetic mapping approach, using seeds from a double haploid population from a cross between broccoli and Chinese kale, produced at three locations. The analysis revealed, besides an effect of the seed production location, a region on linkage group C3 associated with blindness sensitivity. A subsequent dynamic genome-wide transcriptome analysis resulted in the identification of around 3000 differentially expressed genes early after blindness induction. A large number of cell cycle genes were en masse induced early during the development of blindness, whereas shortly after, all were down-regulated. This miss-regulation of core cell cycle genes is accompanied with a strong reduction of cells reaching the DNA replication phase. From the differentially expressed genes, 90 were located in the QTL region C3. Among them are two genes belonging to the MINICHROMOSOMAL MAINTENANCE gene family, known to be involved in DNA replication, a RETINOBLASTOMA-RELATED gene, a key regulator for cell cycle initiation, and several MutS homologs genes, involved in DNA repair. These genes are potential candidates for being involved in the development of blindness in Brassica oleracea sensitive genotypes.