ABSTRACT
Cardiomyocyte maturation is crucial for generating adult cardiomyocytes and the application of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). However, regulation at the cis-regulatory element level and its role in heart disease remain unclear. Alpha-actinin 2 (ACTN2) levels increase during CM maturation. In this study, we investigated a clinically relevant, conserved ACTN2 enhancer's effects on CM maturation using hPSC and mouse models. Heterozygous ACTN2 enhancer deletion led to abnormal CM morphology, reduced function and mitochondrial respiration. Transcriptomic analyses in vitro and in vivo showed disrupted CM maturation and upregulated anabolic mammalian target for rapamycin (mTOR) signaling, promoting senescence and hindering maturation. As confirmation, ACTN2 enhancer deletion induced heat shock protein 90A expression, a chaperone mediating mTOR activation. Conversely, targeting the ACTN2 enhancer via enhancer CRISPR activation (enCRISPRa) promoted hPSC-CM maturation. Our studies reveal the transcriptional enhancer's role in cardiac maturation and disease, offering insights into potentially fine-tuning gene expression to modulate cardiomyocyte physiology.
Subject(s)
Actinin , Cell Differentiation , Enhancer Elements, Genetic , Myocytes, Cardiac , Myocytes, Cardiac/metabolism , Humans , Enhancer Elements, Genetic/genetics , Animals , Actinin/genetics , Actinin/metabolism , Cell Differentiation/genetics , TOR Serine-Threonine Kinases/metabolism , TOR Serine-Threonine Kinases/genetics , HSP90 Heat-Shock Proteins/genetics , HSP90 Heat-Shock Proteins/metabolism , Signal Transduction/genetics , Mice , Transcription, Genetic , Gene Expression Regulation, Developmental , Cell Line , PhenotypeABSTRACT
Blood vessels serve as intermediate conduits for the extension of sympathetic axons towards target tissues, while also acting as crucial targets for their homeostatic processes encompassing the regulation of temperature, blood pressure, and oxygen availability. How sympathetic axons innervate not only blood vessels but also a wide array of target tissues is not clear. Here we show that in embryonic skin, after the establishment of co-branching between sensory nerves and blood vessels, sympathetic axons invade the skin alongside these sensory nerves and extend their branches towards these blood vessels covered by vascular smooth muscle cells (VSMCs). Our mosaic labeling technique for sympathetic axons shows that collateral branching predominantly mediates the innervation of VSMC-covered blood vessels by sympathetic axons. The expression of nerve growth factor (NGF), previously known to induce collateral axon branching in culture, can be detected in the vascular smooth muscle cell (VSMC)-covered blood vessels, as well as sensory nerves. Indeed, VSMC-specific Ngf knockout leads to a significant decrease of collateral branching of sympathetic axons innervating VSMC-covered blood vessels. These data suggest that VSMC-derived NGF serves as an inductive signal for collateral branching of sympathetic axons innervating blood vessels in the embryonic skin.
Subject(s)
Muscle, Smooth, Vascular , Nerve Growth Factor , Skin , Animals , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/innervation , Nerve Growth Factor/metabolism , Mice , Skin/innervation , Skin/blood supply , Skin/metabolism , Myocytes, Smooth Muscle/metabolism , Axons/metabolism , Axons/physiology , Blood Vessels/embryology , Blood Vessels/innervation , Blood Vessels/metabolism , Sympathetic Nervous System/embryology , Sympathetic Nervous System/physiology , Sympathetic Nervous System/metabolism , Mice, KnockoutABSTRACT
Neurons can regulate the development, pathogenesis, and regeneration of target organs. However, the role of neurons during heart development and regeneration remains unclear. We genetically inhibited sympathetic innervation in vivo, which resulted in heart enlargement with an increase in cardiomyocyte number. Transcriptomic and protein analysis showed down-regulation of the two clock gene homologs Period1/Period2 (Per1/Per2) accompanied by up-regulation of cell cycle genes. Per1/Per2 deletion increased heart size and cardiomyocyte proliferation, recapitulating sympathetic neurondeficient hearts. Conversely, increasing sympathetic activity by norepinephrine treatment induced Per1/Per2 and suppressed cardiomyocyte proliferation. We further found that the two clock genes negatively regulate myocyte mitosis entry through the Wee1 kinase pathway. Our findings demonstrate a previously unknown link between cardiac neurons and clock genes in regulation of cardiomyocyte proliferation and heart size and provide mechanistic insights for developing neuromodulation strategies for cardiac regen5eration.
ABSTRACT
PURPOSE OF REVIEW: Heart development is a meticulously coordinated process that involves the specification of two distinct populations of cardiac progenitor cells, namely the first and the second heart field. Disruption of heart field progenitors can result in congenital heart defects. In this review, we aim to describe the signaling pathways and transcription factors that link heart field development and congenital heart disease. RECENT FINDINGS: Single-cell transcriptomics, lineage-tracing mouse models, and stem cell-based in vitro modeling of cardiogenesis have significantly improved the spatiotemporal characterization of cardiac progenitors. Additionally, novel functional genomic studies have now linked more genetic variants with congenital heart disease. Dysregulation of cardiac progenitor cells causes malformations that can be lethal. Ongoing research will continue to shed light on cardiac morphogenesis and help us better understand and treat patients with congenital heart disease.