Although congenital heart defects (CHDs) represent the most common birth defect, a comprehensive understanding of disease etiology remains unknown. This is further complicated since CHDs can occur in isolation or as a feature of another disorder. Analyzing disorders with associated CHDs provides a powerful platform to identify primary pathogenic mechanisms driving disease. Aberrant localization and expression of cathepsin proteases can perpetuate later-stage heart diseases, but their contribution toward CHDs is unclear. To investigate the contribution of cathepsins during cardiovascular development and congenital disease, we analyzed the pathogenesis of cardiac defects in zebrafish models of the lysosomal storage disorder mucolipidosis II (MLII). MLII is caused by mutations in the GlcNAc-1-phosphotransferase enzyme (Gnptab) that disrupt carbohydrate-dependent sorting of lysosomal enzymes. Without Gnptab, lysosomal hydrolases, including cathepsin proteases, are inappropriately secreted. Analyses of heart development in gnptab-deficient zebrafish show cathepsin K secretion increases its activity, disrupts TGF-β–related signaling, and alters myocardial and valvular formation. Importantly, cathepsin K inhibition restored normal heart and valve development in MLII embryos. Collectively, these data identify mislocalized cathepsin K as an initiator of cardiac disease in this lysosomal disorder and establish cathepsin inhibition as a viable therapeutic strategy.
Po-Nien Lu, Trevor Moreland, Courtney J. Christian, Troy C. Lund, Richard A. Steet, Heather Flanagan-Steet
Atrial fibrillation (AF) is the most common cardiac arrhythmia, yet the molecular signature of the vulnerable atrial substrate is not well understood. Here, we delineated a distinct transcriptional signature in right versus left atrial cardiomyocytes (CMs) at baseline, and identified chamber-specific gene expression changes in patients with history of AF in the setting of end-stage heart failure (AF+HF) that are not present in heart failure alone (HF). We observed that human left atrial (LA) CMs exhibit Notch pathway activation and increased ploidy in AF+HF, but not in HF alone. Transient activation of Notch signaling within adult CMs in a murine genetic model is sufficient to increase ploidy in both atrial chambers. Notch activation within LA CMs generated a transcriptomic fingerprint resembling AF, with dysregulation of transcription factor and ion channel genes including Pitx2, Tbx5, Kcnh2, Kcnq1, and Kcnip2. Notch activation also produced distinct cellular electrophysiologic responses in LA versus RA CMs, prolonging the action potential duration (APD) without altering the upstroke velocity in the LA, and reducing the maximal upstroke velocity without altering the APD in the RA. Our results support a shared human/murine model of increased Notch pathway activity predisposing to AF.
Catherine E. Lipovsky, Jesus Jimenez, Qiusha Guo, Gang Li, Tiankai Yin, Stephanie Hicks, Somya Bhatnagar, Kentaro Takahashi, David M. Zhang, Brittany D. Brumback, Uri Goldsztejn, Rangarajan D. Nadadur, Carlos Perez-Cervantes, Ivan P. Moskowitz, Shaopeng Liu, Bo Zhang, Stacey L. Rentschler
Glaucoma surgeries, such as trabeculectomy, are performed to lower intraocular pressure to reduce risk of vision loss. These surgeries create a new passage in the eye that reroutes the aqueous humor outflow to the subconjunctival space, where the fluid is presumably absorbed by the conjunctival lymphatics. Here, we characterized the development and function of the ocular lymphatics using transgenic lymphatic reporter mice and rats. We found that the limbal and conjunctival lymphatic networks are progressively formed from a primary lymphatic vessel that grows from the nasal-side medial canthus region at birth. This primary lymphatic vessel immediately branches out, invades the limbus and conjunctiva, and bidirectionally encircles the cornea. As a result, the distribution of the ocular lymphatics is significantly polarized toward the nasal side, and the limbal lymphatics are directly connected to the conjunctival lymphatics. New lymphatic sprouts are produced mainly from the nasal-side limbal lymphatics, posing the nasal side of the eye as more responsive to fluid drainage and inflammatory stimuli. Consistent with this polarized distribution of the ocular lymphatics, a higher drainage efficiency was observed in the nasal side than the temporal side of the eye when injected with a fluorescent tracer. In contrast, blood vessels are evenly distributed at the anterior surface of the eyes. Also, we found that these distinct vascular distribution patterns were conserved in human eyes. Together, our study demonstrated that the ocular surface lymphatics are more densely present in the nasal side and uncovered the potential clinical benefits in selecting the nasal side as a glaucoma surgery site to improve fluid drainage.
Yifan Wu, Young Jin Seong, Kin Li, Dongwon Choi, Eunkyung Park, George H. Daghlian, Eunson Jung, Khoa Bui, Luping Zhao, Shrimika Madhavan, Saren Daghlian, Patill Daghlian, Desmond Chin, Il-Taeg Cho, Alex K. Wong, Martin Heur, Sandy Zhang-Nunes, James C. Tan, Masatsugu Ema, Tina T. Wong, Alex S. Huang, Young-Kwon Hong
During the growth of lymphatic vessels (lymphangiogenesis), lymphatic endothelial cells (LECs) at the growing front sprout by forming filopodia. Those tip cells are not exposed to circulating lymph, as they are not lumenized. In contrast, LECs that trail the growing front are exposed to shear stress, become quiescent and remodel into stable vessels. The mechanisms that coordinate the opposed activities of lymphatic sprouting and maturation remain poorly understood. Here we show that the canonical tip cell marker Delta-Like 4 (DLL4) promotes sprouting lymphangiogenesis by enhancing Vascular Endothelial Growth Factor C (VEGF-C) /VEGF Receptor 3 (VEGFR3) signaling. However, in lumenized lymphatic vessels laminar shear stress (LSS) inhibits the expression of DLL4, as well as additional tip cell markers. Paradoxically, LSS also upregulates VEGF-C/VEGFR3 signaling in LECs, but sphingosine 1-phosphate (S1P) receptor 1 (S1PR1) activity antagonizes LSS-mediated VEGF-C signaling to promote lymphatic vascular quiescence. Correspondingly, S1pr1 loss in LECs induced lymphatic vascular hypersprouting and hyperbranching, which could be rescued by reducing Vegfr3 gene dosage in vivo. In addition, S1PR1 regulates lymphatic vessel maturation by inhibiting RhoA activity to promote membrane localization of the tight junction molecule Claudin-5. Our findings suggest a new paradigm in which LSS induces quiescence and promotes the survival of LECs by downregulating DLL4 and enhancing VEGF-C signaling, respectively. S1PR1 dampens LSS/VEGF-C signaling, thereby preventing sprouting from quiescent lymphatic vessels. These results also highlight the distinct roles that S1PR1 and DLL4 play in LECs when compared to their known roles in the blood vasculature.
Xin Geng, Keisuke Yanagida, Racheal G. Akwii, Dongwon Choi, Lijuan Chen, YenChun Ho, Boksik Cha, Md. Riaj Mahamud, Karen Berman de Ruiz, Hirotake Ichise, Hong Chen, Joshua Wythe, Constantinos M. Mikelis, Timothy Hla, R. Sathish Srinivasan
The impact of transient ischemic-hypoxemic insults on the developing fetal brain is poorly understood despite evidence suggesting an association with neurodevelopmental disorders such as schizophrenia and autism. To address this, we designed an aberrant uterine hypercontractility paradigm with oxytocin to better assess the consequences of acute, but transient, placental ischemia-hypoxemia in term pregnant rats. Using MRI, we confirmed that oxytocin-induced aberrant uterine hypercontractility substantially compromised uteroplacental perfusion. This was supported by the observation of oxidative stress and increased lactate concentration in the fetal brain. Genes related to oxidative stress pathways were significantly upregulated in male, but not female, offspring 1 hour after oxytocin-induced placental ischemia-hypoxemia. Persistent upregulation of select mitochondrial electron transport chain complex proteins in the anterior cingulate cortex of adolescent male offspring suggested that this sex-specific effect was enduring. Functionally, offspring exposed to oxytocin-induced uterine hypercontractility showed male-specific abnormalities in social behavior with associated region-specific changes in gene expression and functional cortical connectivity. Our findings, therefore, indicate that even transient but severe placental ischemia-hypoxemia could be detrimental to the developing brain and point to a possible mitochondrial link between intrauterine asphyxia and neurodevelopmental disorders.
Arvind Palanisamy, Tusar Giri, Jia Jiang, Annie Bice, James D. Quirk, Sara B. Conyers, Susan E. Maloney, Nandini Raghuraman, Adam Q. Bauer, Joel R. Garbow, David F. Wozniak
Insufficient O2 supply is frequently associated with fetal growth restriction (FGR), a leading cause of perinatal mortality and morbidity. Although the erythrocyte is the most abundant and only cell type to deliver O2 in our body, its function and regulatory mechanism in FGR remain unknown. Here, we report that genetic ablation of mouse erythrocyte equilibrative nucleoside transporter 1 (eENT1) in dams, but not placentas or fetuses, results in FGR. Unbiased high-throughput metabolic profiling coupled with in vitro and in vivo flux analyses with isotopically labeled tracers led us to discover that maternal eENT1–dependent adenosine uptake is critical in activating AMPK by controlling the AMP/ATP ratio and its downstream target, bisphosphoglycerate mutase (BPGM); in turn, BPGM mediates 2,3-BPG production, which enhances O2 delivery to maintain placental oxygenation. Mechanistically and functionally, we revealed that genetic ablation of maternal eENT1 increases placental HIF-1α; preferentially reduces placental large neutral aa transporter 1 (LAT1) expression, activity, and aa supply; and induces FGR. Translationally, we revealed that elevated HIF-1α directly reduces LAT1 gene expression in cultured human trophoblasts. We demonstrate the importance and molecular insight of maternal eENT1 in fetal growth and open up potentially new diagnostic and therapeutic possibilities for FGR.
Seisuke Sayama, Anren Song, Benjamin C. Brown, Jacob Couturier, Xiaoli Cai, Ping Xu, Changhan Chen, Yangxi Zheng, Takayuki Iriyama, Baha Sibai, Monica Longo, Rodney E. Kellems, Angelo D’Alessandro, Yang Xia
Refractory neonatal seizures do not respond to first-line anti-seizure medications (ASMs) like phenobarbital (PB), a positive allosteric modulator for GABAA receptors. GABAA receptor-mediated inhibition is dependent upon electroneutral cation-chloride transporter KCC2 which mediates neuronal chloride extrusion and its age-dependent increase, postnatally shifts GABAergic signaling from depolarizing to hyperpolarizing. BDNF-TrkB activation following excitotoxic injury recruits downstream targets like PLCγ1, leading to KCC2 hypofunction. Here, the anti-seizure efficacy of TrkB agonists LM22A-4, HIOC, and Deoxygedunin (DG), on PB-refractory seizures, and post-ischemic TrkB-pathway activation was investigated in a mouse model (CD-1, P7) of refractory neonatal seizures. LM, a BDNF loop II mimetic, rescued PB-refractory seizures in a sexually dimorphic manner. Efficacy was associated with a significant reduction in the post-ischemic phosphorylation of TrkB at Y816, a site known to mediate post-ischemic KCC2 hypofunction via PLCγ1 activation. LM rescued ischemia-induced pKCC2-S940 dephosphorylation, preserving its membrane stability. Full TrkB agonists HIOC and DG similarly rescued PB-refractoriness. Chemogenetic inactivation of TrkB significantly reduced post-ischemic neonatal seizure burdens at P7. Sex differences identified in developmental expression profiles of TrkB and KCC2 may underlie the sexually dimorphic efficacy of LM. These results support a novel role for the TrkB receptor in the emergence of age-dependent refractory neonatal seizures.
Pavel A. Kipnis, Brennan J. Sullivan, Brandon M. Carter, Shilpa Kadam
Hydrocephalus is characterized by abnormal accumulation of cerebrospinal fluid (CSF) in the ventricular cavity. The circulation of CSF in brain ventricles is controlled by the coordinated beating of motile cilia at the surface of ependymal cells (ECs). Here we show that MT1-MMP is highly expressed in olfactory bulb, rostral migratory stream, and ventricular system. Mice deficient for Membrane type-1-MMP (MT1-MMP) develop typical phenotypes observed in hydrocephalus such as dome-shaped skull, dilated ventricles, corpus callosum agenesis and astrocyte hypertrophy during the first two weeks of postnatal development. MT1-MMP deficient mice exhibits reduced and disorganized motile cilia with the impaired maturation of ECs, leading to abnormal CSF flow. Consistent with the defects in motile cilia morphogenesis, the expressions of pro-multiciliogenic genes are significantly decreased with a concomitant hyper-activation of Notch signaling in the wall of lateral ventricles in Mmp14-/- brains. Inhibition of Notch signaling by γ-secretase inhibitor restores ciliogenesis in Mmp14-/- ECs. Taken together, these data suggest that MT1-MMP is required for ciliogenesis and ependymal cell maturation by suppressing Notch signaling during early brain development. Our findings implicate that MT1-MMP is critical for early brain development and loss of MT1-MMP activity gives rise to hydrocephalus.
Zhixin Jiang, Jin Zhou, Xin Qin, Huiling Zheng, Bo Gao, Xin-guang Liu, Guoxiang Jin, Zhongjun Zhou
Decades ago, investigators reported that mice lacking DLX1 and DLX2, transcription factors expressed in the enteric nervous system (ENS), die with possible bowel motility problems. These problems were never fully elucidated. We found that mice lacking DLX1 and DLX2 (Dlx1/2-/- mice) had slower small bowel transit and reduced or absent neurally-mediated contraction complexes. In contrast, small bowel motility seemed normal in adult mice lacking DLX1 (Dlx1-/-). Even with detailed anatomic studies, we found no defects in ENS precursor migration, or neuron and glia density, in Dlx1/2-/- or Dlx1-/- mice. However, RNA sequencing of Dlx1/2-/- ENS revealed dysregulation of many genes, including vasoactive intestinal peptide (Vip). Our study reveals a novel connection between Dlx genes and Vip and highlights the observation that dangerous bowel motility problems can occur in the absence of easily identifiable ENS structural defects. These findings may be relevant for disorders like chronic intestinal pseudo-obstruction (CIPO) syndrome.
Christina M. Wright, James P. Garifallou, Sabine Schneider, Heather L. Mentch, Deepika R. Kothakapa, Beth A. Maguire, Robert O. Heuckeroth
Deterioration or inborn malformations of the cardiac conduction system (CCS) interfere with proper impulse propagation in the heart and may lead to sudden cardiac death or heart failure. Patients afflicted with arrhythmia depend on antiarrhythmic medication or invasive therapy, such as pacemaker implantation. An ideal way to treat these patients would be CCS tissue restoration. This, however, requires precise knowledge regarding the molecular mechanisms underlying CCS development. Here, we aimed to identify regulators of CCS development. We performed a compound screen in zebrafish embryos and identified tolterodine, a muscarinic receptor antagonist, as a modifier of CCS development. Tolterodine provoked a lower heart rate, pericardiac edema, and arrhythmia. Blockade of muscarinic M3, but not M2, receptors induced transcriptional changes leading to amplification of sinoatrial cells and loss of atrioventricular identity. Transcriptome data from an engineered human heart muscle model provided additional evidence for the contribution of muscarinic M3 receptors during cardiac progenitor specification and differentiation. Taken together, we found that muscarinic M3 receptors control the CCS already before the heart becomes innervated. Our data indicate that muscarinic receptors maintain a delicate balance between the developing sinoatrial node and the atrioventricular canal, which is probably required to prevent the development of arrhythmia.
Martina S. Burczyk, Martin D. Burkhalter, Teresa Casar Tena, Laurel A. Grisanti, Michael Kauk, Sabrina Matysik, Cornelia Donow, Monika Kustermann, Melanie Rothe, Yinghong Cui, Farah Raad, Svenja Laue, Allessandra Moretti, Wolfram-H. Zimmermann, Jürgen Wess, Michael Kühl, Carsten Hoffmann, Douglas G. Tilley, Melanie Philipp
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