Proliferation and Maturation: Janus and the Art of Cardiac Tissue Engineering.

During cardiac development and morphogenesis, cardiac progenitor cells differentiate into cardiomyocytes that expand in number and size to generate the fully formed heart. Much is known about the factors that regulate initial differentiation of cardiomyocytes, and there is ongoing research to identify how these fetal and immature cardiomyocytes develop into fully functioning, mature cells. Accumulating evidence indicates that maturation limits proliferation and conversely proliferation occurs rarely in cardiomyocytes of the adult myocardium. We term this oppositional interplay the proliferation-maturation dichotomy. Here we review the factors that are involved in this interplay and discuss how a better understanding of the proliferation-maturation dichotomy could advance the utility of human induced pluripotent stem cell-derived cardiomyocytes for modeling in 3-dimensional engineered cardiac tissues to obtain truly adult-level function.

The E3 ubiquitin ligase Nedd4/Nedd4L is directly regulated by microRNA 1.

miR-1 is a small noncoding RNA molecule that modulates gene expression in heart and skeletal muscle. Loss of Drosophila miR-1 produces defects in somatic muscle and embryonic heart development, which have been partly attributed to miR-1 directly targeting Delta to decrease Notch signaling. Here, we show that overexpression of miR-1 in the fly wing can paradoxically increase Notch activity independently of its effects on Delta. Analyses of potential miR-1 targets revealed that miR-1 directly regulates the 3'UTR of the E3 ubiquitin ligase Nedd4 Analysis of embryonic and adult fly heart revealed that the Nedd4 protein regulates heart development in Drosophila Larval fly hearts overexpressing miR-1 have profound defects in actin filament organization that are partially rescued by concurrent overexpression of Nedd4. These results indicate that miR-1 and Nedd4 act together in the formation and actin-dependent patterning of the fly heart. Importantly, we have found that the biochemical and genetic relationship between miR-1 and the mammalian ortholog Nedd4-like (Nedd4l) is evolutionarily conserved in the mammalian heart, potentially indicating a role for Nedd4L in mammalian postnatal maturation. Thus, miR-1-mediated regulation of Nedd4/Nedd4L expression may serve to broadly modulate the trafficking or degradation of Nedd4/Nedd4L substrates in the heart.

A disrupted compartment boundary underlies abnormal cardiac patterning and congenital heart defects.

Failure of septation of the interventricular septum (IVS) is the most common congenital heart defect (CHD), but mechanisms for patterning the IVS are largely unknown. We show that a Tbx5+/Mef2cAHF+ progenitor lineage forms a compartment boundary bisecting the IVS. This coordinated population originates at a first- and second heart field interface, subsequently forming a morphogenetic nexus. Ablation of Tbx5+/Mef2cAHF+ progenitors cause IVS disorganization, right ventricular hypoplasia and mixing of IVS lineages. Reduced dosage of the CHD transcription factor TBX5 disrupts boundary position and integrity, resulting in ventricular septation defects (VSDs) and patterning defects, including Slit2 and Ntn1 misexpression. Reducing NTN1 dosage partly rescues cardiac defects in Tbx5 mutant embryos. Loss of Slit2 or Ntn1 causes VSDs and perturbed septal lineage distributions. Thus, we identify essential cues that direct progenitors to pattern a compartment boundary for proper cardiac septation, revealing new mechanisms for cardiac birth defects.

Metabolic Changes during In Vivo Maturation of PSC-Derived Skeletal Myogenic Progenitors.

In vitro-generated pluripotent stem cell (PSC)-derived Pax3-induced (iPax3) myogenic progenitors display an embryonic transcriptional signature, but upon engraftment, the profile of re-isolated iPax3 donor-derived satellite cells changes toward similarity with postnatal satellite cells, suggesting that engrafted PSC-derived myogenic cells remodel their transcriptional signature upon interaction within the adult muscle environment. Here, we show that engrafted myogenic progenitors also remodel their metabolic state. Assessment of oxygen consumption revealed that exposure to the adult muscle environment promotes overt changes in mitochondrial bioenergetics, as shown by the substantial suppression of energy requirements in re-isolated iPax3 donor-derived satellite cells compared to their in vitro-generated progenitors. Mass spectrometry-based metabolomic profiling further confirmed the relationship of engrafted iPax3 donor-derived cells to adult satellite cells. The fact that in vitro-generated myogenic progenitors remodel their bioenergetic signature upon in vivo exposure to the adult muscle environment may have important implications for therapeutic applications.

Stimulation of Cardiomyocyte Proliferation Is Dependent on Species and Level of Maturation.

The heart is one of the least regenerative organs. This is in large part due to the inability of adult mammalian cardiomyocytes to proliferate and divide. In recent years, a number of small molecules and molecular targets have been identified to stimulate cardiomyocyte proliferation, including p38 inhibition, YAP-Tead activation, fibroblast growth factor 1 and Neuregulin 1. Despite these exciting initial findings, a therapeutic approach to enhance cardiomyocyte proliferation in vivo is still lacking. We hypothesized that a more comprehensive in vitro validation using live-cell imaging and assessment of the proliferative effects on various cardiomyocyte sources might identify the most potent proliferative stimuli. Here, we used previously published stimuli to determine their proliferative effect on cardiomyocytes from different species and isolated from different developmental timepoints. Although all stimuli enhanced DNA synthesis and Histone H3 phosphorylation in neonatal rat ventricular cardiomyocytes to similar degrees, these effects varied substantially in mouse cardiomyocytes and human iPSC-derived cardiomyocytes. Our results highlight p21 inhibition and Yap-Tead activation as potent proliferative strategies to induce cultured cardiomyocyte cell cycle activity across mouse, rat and human cardiomyocytes.

Dual inhibition of MAPK and PI3K/AKT pathways enhances maturation of human iPSC-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide great opportunities for mechanistic dissection of human cardiac pathophysiology; however, hiPSC-CMs remain immature relative to the adult heart. To identify novel signaling pathways driving the maturation process during heart development, we analyzed published transcriptional and epigenetic datasets from hiPSC-CMs and prenatal and postnatal human hearts. These analyses revealed that several components of the MAPK and PI3K-AKT pathways are downregulated in the postnatal heart. Here, we show that dual inhibition of these pathways for only 5 days significantly enhances the maturation of day 30 hiPSC-CMs in many domains: hypertrophy, multinucleation, metabolism, T-tubule density, calcium handling, and electrophysiology, many equivalent to day 60 hiPSC-CMs. These data indicate that the MAPK/PI3K/AKT pathways are involved in cardiomyocyte maturation and provide proof of concept for the manipulation of key signaling pathways for optimal hiPSC-CM maturation, a critical aspect of faithful in vitro modeling of cardiac pathologies and subsequent drug discovery.

Genomic Safe Harbor Expression of PAX7 for the Generation of Engraftable Myogenic Progenitors.

Inducible expression of PAX7 in differentiating pluripotent stem cells (PSCs) allows massively scalable generation of human myogenic progenitors, which upon transplantation into dystrophic muscles give rise to donor-derived myofibers and satellite cells. Therefore, PSC-derived PAX7+ myogenic progenitors represent an attractive therapeutic approach to promote muscle regeneration. Work to date has used lentiviral vectors (LVs) that randomly integrate inducible PAX7 transgenes. Here, we investigated whether equivalent induction of the myogenic program could be achieved by targeting the PAX7 transgene into genomic safe harbor (GSH) sites. Across multiple PSC lines, we find that this approach consistently generates expandable myogenic progenitors in vitro, although scalability of expansion is moderately reduced compared with the LV approach. Importantly, transplantation of GSH-targeted myogenic progenitors produces robust engraftment, comparable with LV counterparts. These findings provide proof of concept for the use of GSH targeting as a potential alternative approach to generate therapeutic PSC-derived myogenic progenitors for clinical applications.

Modeling Human TBX5 Haploinsufficiency Predicts Regulatory Networks for Congenital Heart Disease.

Haploinsufficiency of transcriptional regulators causes human congenital heart disease (CHD); however, the underlying CHD gene regulatory network (GRN) imbalances are unknown. Here, we define transcriptional consequences of reduced dosage of the CHD transcription factor, TBX5, in individual cells during cardiomyocyte differentiation from human induced pluripotent stem cells (iPSCs). We discovered highly sensitive dysregulation of TBX5-dependent pathways-including lineage decisions and genes associated with heart development, cardiomyocyte function, and CHD genetics-in discrete subpopulations of cardiomyocytes. Spatial transcriptomic mapping revealed chamber-restricted expression for many TBX5-sensitive transcripts. GRN analysis indicated that cardiac network stability, including vulnerable CHD-linked nodes, is sensitive to TBX5 dosage. A GRN-predicted genetic interaction between Tbx5 and Mef2c, manifesting as ventricular septation defects, was validated in mice. These results demonstrate exquisite and diverse sensitivity to TBX5 dosage in heterogeneous subsets of iPSC-derived cardiomyocytes and predicts candidate GRNs for human CHDs, with implications for quantitative transcriptional regulation in disease.

A second X chromosome contributes to resilience in a mouse model of Alzheimer's disease.

A major sex difference in Alzheimer's disease (AD) is that men with the disease die earlier than do women. In aging and preclinical AD, men also show more cognitive deficits. Here, we show that the X chromosome affects AD-related vulnerability in mice expressing the human amyloid precursor protein (hAPP), a model of AD. XY-hAPP mice genetically modified to develop testicles or ovaries showed worse mortality and deficits than did XX-hAPP mice with either gonad, indicating a sex chromosome effect. To dissect whether the absence of a second X chromosome or the presence of a Y chromosome conferred a disadvantage on male mice, we varied sex chromosome dosage. With or without a Y chromosome, hAPP mice with one X chromosome showed worse mortality and deficits than did those with two X chromosomes. Thus, adding a second X chromosome conferred resilience to XY males and XO females. In addition, the Y chromosome, its sex-determining region Y gene (Sry), or testicular development modified mortality in hAPP mice with one X chromosome such that XY males with testicles survived longer than did XY or XO females with ovaries. Furthermore, a second X chromosome conferred resilience potentially through the candidate gene Kdm6a, which does not undergo X-linked inactivation. In humans, genetic variation in KDM6A was linked to higher brain expression and associated with less cognitive decline in aging and preclinical AD, suggesting its relevance to human brain health. Our study suggests a potential role for sex chromosomes in modulating disease vulnerability related to AD.

Peripheral Elevation of a Klotho Fragment Enhances Brain Function and Resilience in Young, Aging, and α-Synuclein Transgenic Mice.

Cognitive dysfunction and decreased mobility from aging and neurodegenerative conditions, such as Parkinson and Alzheimer diseases, are major biomedical challenges in need of more effective therapies. Increasing brain resilience may represent a new treatment strategy. Klotho, a longevity factor, enhances cognition when genetically and broadly overexpressed in its full, wild-type form over the mouse lifespan. Whether acute klotho treatment can rapidly enhance cognitive and motor functions or induce resilience is a gap in our knowledge of its therapeutic potential. Here, we show that an α-klotho protein fragment (αKL-F), administered peripherally, surprisingly induced cognitive enhancement and neural resilience despite impermeability to the blood-brain barrier in young, aging, and transgenic α-synuclein mice. αKL-F treatment induced cleavage of the NMDAR subunit GluN2B and also enhanced NMDAR-dependent synaptic plasticity. GluN2B blockade abolished αKL-F-mediated effects. Peripheral αKL-F treatment is sufficient to induce neural enhancement and resilience in mice and may prove therapeutic in humans.