ABSTRACT
Helper T effector cytokines implicated in asthma modulate the contractility of human airway smooth muscle (HASM) cells. We have reported recently that a profibrotic cytokine, transforming growth factor (TGF)-ß1, induces HASM cell shortening and airway hyperresponsiveness. Here, we assessed whether TGF-ß1 affects the ability of HASM cells to relax in response to ß2-agonists, a mainstay treatment for airway hyperresponsiveness in asthma. Overnight TGF-ß1 treatment significantly impaired isoproterenol (ISO)-induced relaxation of carbachol-stimulated, isolated HASM cells. This single-cell mechanical hyporesponsiveness to ISO was corroborated by sustained increases in myosin light chain phosphorylation. In TGF-ß1-treated HASM cells, ISO evoked markedly lower levels of intracellular cAMP. These attenuated cAMP levels were, in turn, restored with pharmacological and siRNA inhibition of phosphodiesterase 4 and Smad3, respectively. Most strikingly, TGF-ß1 selectively induced phosphodiesterase 4D gene expression in HASM cells in a Smad2/3-dependent manner. Together, these data suggest that TGF-ß1 decreases HASM cell ß2-agonist relaxation responses by modulating intracellular cAMP levels via a Smad2/3-dependent mechanism. Our findings further define the mechanisms underlying ß2-agonist hyporesponsiveness in asthma, and suggest TGF-ß1 as a potential therapeutic target to decrease asthma exacerbations in severe and treatment-resistant asthma.
Subject(s)
Asthma/physiopathology , Muscle, Smooth/metabolism , Smad2 Protein/metabolism , Smad3 Protein/metabolism , Transforming Growth Factor beta1/metabolism , Transforming Growth Factor beta2/agonists , Asthma/drug therapy , Asthma/metabolism , Bronchodilator Agents/pharmacology , Carbachol/pharmacology , Cyclic AMP/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 4/metabolism , Cytokines/metabolism , Gene Expression Regulation , Humans , Isoproterenol/pharmacology , Lung/metabolism , Muscle, Smooth/drug effects , Myosin Light Chains/metabolism , Phosphorylation , RNA, Small Interfering/metabolism , Trachea/drug effects , Trachea/metabolism , Transforming Growth Factor beta2/metabolismABSTRACT
We recently demonstrated that blue light induces vasorelaxation in the systemic mouse circulation, a phenomenon mediated by the nonvisual G protein-coupled receptor melanopsin (Opsin 4; Opn4). Here we tested the hypothesis that nonvisual opsins mediate photorelaxation in the pulmonary circulation. We discovered Opsin 3 (Opn3), Opn4, and G protein-coupled receptor kinase 2 (GRK2) in rat pulmonary arteries (PAs) and in pulmonary arterial smooth muscle cells (PASMCs), where the opsins interact directly with GRK2, as demonstrated with a proximity ligation assay. Light elicited an intensity-dependent relaxation of PAs preconstricted with phenylephrine (PE), with a maximum response between 400 and 460 nm (blue light). Wavelength-specific photorelaxation was attenuated in PAs from Opn4-/- mice and further reduced following shRNA-mediated knockdown of Opn3. Inhibition of GRK2 amplified the response and prevented physiological desensitization to repeated light exposure. Blue light also prevented PE-induced constriction in isolated PAs, decreased basal tone, ablated PE-induced single-cell contraction of PASMCs, and reversed PE-induced depolarization in PASMCs when GRK2 was inhibited. The photorelaxation response was modulated by soluble guanylyl cyclase but not by protein kinase G or nitric oxide. Most importantly, blue light induced significant vasorelaxation of PAs from rats with chronic pulmonary hypertension and effectively lowered pulmonary arterial pressure in isolated intact perfused rat lungs subjected to acute hypoxia. These findings show that functional Opn3 and Opn4 in PAs represent an endogenous "optogenetic system" that mediates photorelaxation in the pulmonary vasculature. Phototherapy in conjunction with GRK2 inhibition could therefore provide an alternative treatment strategy for pulmonary vasoconstrictive disorders.
Subject(s)
G-Protein-Coupled Receptor Kinase 2/antagonists & inhibitors , Hypertension, Pulmonary/radiotherapy , Phototherapy , Pulmonary Artery/radiation effects , Rod Opsins/physiology , Vasodilation/radiation effects , Animals , Cells, Cultured , G-Protein-Coupled Receptor Kinase 2/genetics , G-Protein-Coupled Receptor Kinase 2/metabolism , Hypertension, Pulmonary/etiology , Hypertension, Pulmonary/pathology , Hypoxia/complications , Light , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/radiation effects , Nitric Oxide/metabolism , Pulmonary Artery/cytology , Pulmonary Artery/metabolism , Rats , Rats, Sprague-Dawley , Rats, Wistar , Soluble Guanylyl Cyclase/genetics , Soluble Guanylyl Cyclase/metabolism , Vasodilation/physiologyABSTRACT
Cardiomyocytes undergo significant structural and functional changes after birth, and these fundamental processes are essential for the heart to pump blood to the growing body. However, due to the challenges of isolating single postnatal/adult myocytes, how individual newborn cardiomyocytes acquire multiple aspects of the mature phenotype remains poorly understood. Here we implement large-particle sorting and analyze single myocytes from neonatal to adult hearts. Early myocytes exhibit wide-ranging transcriptomic and size heterogeneity that is maintained until adulthood with a continuous transcriptomic shift. Gene regulatory network analysis followed by mosaic gene deletion reveals that peroxisome proliferator-activated receptor coactivator-1 signaling, which is active in vivo but inactive in pluripotent stem cell-derived cardiomyocytes, mediates the shift. This signaling simultaneously regulates key aspects of cardiomyocyte maturation through previously unrecognized proteins, including YAP1 and SF3B2. Our study provides a single-cell roadmap of heterogeneous transitions coupled to cellular features and identifies a multifaceted regulator controlling cardiomyocyte maturation.