The severity of intestinal disease associated with Cystic Fibrosis (CF) is variable in the patient population and this variability is partially conferred by the influence of modifier genes. ...Genome-wide association studies have identified
an electrogenic amino acid transporter, as a genetic modifier of CF-associated meconium ileus. The purpose of the current work was to determine the biological role of
by disrupting its expression in CF mice bearing the major mutation, F508del. We found that disruption of
worsened the intestinal fluid secretion defect, characteristic of these mice. In vitro studies of mouse intestinal organoids revealed that exacerbation of the primary defect was associated with reduced arginine uptake across the apical membrane, with aberrant nitric oxide and cyclic GMP-mediated regulation of the major CF-causing mutant protein. Together, these studies highlight the role of this apical transporter in modifying cellular nitric oxide levels, residual function of the major CF mutant and potentially, its promise as a therapeutic target.
SLC6A14-mediated l-arginine transport has been shown to augment the residual anion channel activity of the major mutant, F508del-CFTR, in the murine gastrointestinal tract. It is not yet known if ...this transporter augments residual and pharmacological corrected F508del-CFTR in primary airway epithelia. We sought to determine the role of l-arginine uptake via SLC6A14 in modifying F508del-CFTR channel activity in airway cells from patients with cystic fibrosis (CF). Human bronchial epithelial (HBE) cells from lung explants of patients without CF (HBE) and those with CF (CF-HBE) were used for H
-flux, airway surface liquid, and Ussing chamber studies. We used α-methyltryptophan as a specific inhibitor for SLC6A14. CFBE41o
, a commonly used CF airway cell line, was employed for studying the mechanism of the functional interaction between SLC6A14 and F508del-CFTR. SLC6A14 is functionally expressed in CF-HBE cells. l-arginine uptake via SLC6A14 augmented F508del-CFTR function at baseline and after treatment with lumacaftor. SLC6A14-mediated l-arginine uptake also increased the airway surface liquid in CF-HBE cells. Using CFBE41o cells, we showed that the positive SLC6A14 effect was mainly dependent on the nitric oxide (NO) synthase activity, nitrogen oxides, including NO, and phosphorylation by protein kinase G. These finding were confirmed in CF-HBE, as inducible NO synthase inhibition abrogated the functional interaction between SLC6A14 and pharmacological corrected F508del-CFTR. In summary, SLC6A14-mediated l-arginine transport augments residual F508del-CFTR channel function via a noncanonical, NO pathway. This effect is enhanced with increasing pharmacological rescue of F508del-CFTR to the membrane. The current study demonstrates how endogenous pathways can be used for the development of companion therapy in CF.
In the current study we examined several proteomic- and RNA-Seq-based datasets of cardiac-enriched, cell-surface and membrane-associated proteins in human fetal and mouse neonatal ventricular ...cardiomyocytes. By integrating available microarray and tissue expression profiles with MGI phenotypic analysis, we identified 173 membrane-associated proteins that are cardiac-enriched, conserved amongst eukaryotic species, and have not yet been linked to a 'cardiac' Phenotype-Ontology. To highlight the utility of this dataset, we selected several proteins to investigate more carefully, including FAM162A, MCT1, and COX20, to show cardiac enrichment, subcellular distribution and expression patterns in disease. We performed three-dimensional confocal imaging analysis to validate subcellular localization and expression in adult mouse ventricular cardiomyocytes. FAM162A, MCT1, and COX20 were expressed differentially at the transcriptomic and proteomic levels in multiple models of mouse and human heart diseases and may represent potential diagnostic and therapeutic targets for human dilated and ischemic cardiomyopathies. Altogether, we believe this comprehensive cardiomyocyte membrane proteome dataset will prove instrumental to future investigations aimed at characterizing heart disease markers and/or therapeutic targets for heart failure.
The combination therapy of lumacaftor and ivacaftor (Orkambi®) is approved for patients bearing the major cystic fibrosis (CF) mutation: ΔF508. It has been predicted that Orkambi® could treat ...patients with rarer mutations of similar “theratype”; however, a standardized approach confirming efficacy in these cohorts has not been reported. Here, we demonstrate that patients bearing the rare mutation: c.3700 A>G, causing protein misprocessing and altered channel function—similar to ΔF508‐CFTR, are unlikely to yield a robust Orkambi® response. While in silico and biochemical studies confirmed that this mutation could be corrected and potentiated by lumacaftor and ivacaftor, respectively, this combination led to a minor in vitro response in patient‐derived tissue. A CRISPR/Cas9‐edited bronchial epithelial cell line bearing this mutation enabled studies showing that an “amplifier” compound, effective in increasing the levels of immature CFTR protein, augmented the Orkambi® response. Importantly, this “amplifier” effect was recapitulated in patient‐derived nasal cultures—providing the first evidence for its efficacy in augmenting Orkambi® in tissues harboring a rare CF‐causing mutation. We propose that this multi‐disciplinary approach, including creation of CRISPR/Cas9‐edited cells to profile modulators together with validation using primary tissue, will facilitate therapy development for patients with rare CF mutations.
Synopsis
Potential strategies for improving function in a rare CF‐causing mutation based on a CRISPR/Cas9‐edited bronchial cell line and patient‐derived nasal cultures.
Molecular dynamic simulations predicted the consequences of the rare mutation c.3700 A>G on CFTR protein (ΔI1234_R1239‐CFTR) structure.
Misprocessing and altered function of ΔI1234_R1239‐CFTR can be partially ameliorated by small molecule modulators of ΔF508‐CFTR.
A CRISPR/Cas9‐edited HBE cell line recapitulates the endogenous expression of ΔI1234_R1239‐CFTR and response to ΔF508‐CFTR modulators.
A novel small molecule amplifier (PTI‐CH) improves the effect of the corrector (VX‐809) and potentiator (VX‐770) on ΔI1234_R1239‐CFTR in CRISPR/Cas9‐engineered and patient‐specific tissues.
Potential strategies for improving function in a rare CF‐causing mutation based on a CRISPR/Cas9‐edited bronchial cell line and patient‐derived nasal cultures.
The intercalated disc (ICD) is a unique membrane structure that is indispensable to normal heart function, yet its structural organization is not completely understood. Previously, we showed that the ...ICD-bound transmembrane protein 65 (Tmem65) was required for connexin43 (Cx43) localization and function in cultured mouse neonatal cardiomyocytes. Here, we investigate the functional and cellular effects of Tmem65 reductions on the myocardium in a mouse model by injecting CD1 mouse pups (3-7 days after birth) with recombinant adeno-associated virus 9 (rAAV9) harboring Tmem65 shRNA, which reduces Tmem65 expression by 90% in mouse ventricles compared to scrambled shRNA injection. Tmem65 knockdown (KD) results in increased mortality which is accompanied by eccentric hypertrophic cardiomyopathy within 3 weeks of injection and progression to dilated cardiomyopathy with severe cardiac fibrosis by 7 weeks post-injection. Tmem65 KD hearts display depressed hemodynamics as measured echocardiographically as well as slowed conduction in optical recording accompanied by prolonged PR intervals and QRS duration in electrocardiograms. Immunoprecipitation and super-resolution microscopy demonstrate a physical interaction between Tmem65 and sodium channel β subunit (β1) in mouse hearts and this interaction appears to be required for both the establishment of perinexal nanodomain structure and the localization of both voltage-gated sodium channel 1.5 (NaV1.5) and Cx43 to ICDs. Despite the loss of NaV1.5 at ICDs, whole-cell patch clamp electrophysiology did not reveal reductions in Na
currents but did show reduced Ca
and K
currents in Tmem65 KD cardiomyocytes in comparison to control cells. We conclude that disrupting Tmem65 function results in impaired ICD structure, abnormal cardiac electrophysiology, and ultimately cardiomyopathy.
Cell lineage conversion of fibroblasts to specialized cell types through transdifferentiation may provide a fast and alternative cell source for regenerative medicine. Here we show that transient ...transduction of fibroblasts with the four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) in addition to the early lung transcription factor Nkx2-1 (also known as Ttf1), followed by directed differentiation of the cells, can convert mouse embryonic and human adult dermal fibroblasts into induced lung-like epithelial cells (iLEC). These iLEC differentiate into multiple lung cell types in air liquid interface cultures, repopulate decellularized rat lung scaffolds, and form lung epithelia composed of Ciliated, Goblet, Basal, and Club cells after transplantation into immune-compromised mice. As proof-of-concept, differentiated human iLEC harboring the Cystic Fibrosis mutation dF508 demonstrated pharmacological rescue of CFTR function using the combination of lumacaftor and ivacaftor. Overall, this is a promising alternative approach for generation of patient-specific lung-like progenitors to study lung function, disease and future regeneration strategies.
For those people with cystic fibrosis carrying rare CFTR mutations not responding to currently available therapies, there is an unmet need for relevant tissue models for therapy development. Here, we ...describe a new testing platform that employs patient-specific induced pluripotent stem cells (iPSCs) differentiated to lung progenitor cells that can be studied using a dynamic, high-throughput fluorescence-based assay of CFTR channel activity. Our proof-of-concept studies support the potential use of this platform, together with a Canadian bioresource that contains iPSC lines and matched nasal cultures from people with rare mutations, to advance patient-oriented therapy development. Interventions identified in the high-throughput, stem cell-based model and validated in primary nasal cultures from the same person have the potential to be advanced as therapies.
•A Canadian resource (CFIT) has CF donor-matched iPSCs and nasal epithelial cells•Lung progenitor cells (LPCs) differentiated from iPSCs express CFTR•LPCs from people with rare CFTR mutations enable high-throughput therapy testing•Matching nasal cultures can validate patient-specific drug responses in LPCs
Bear and colleagues show that lung progenitor cells (LPCs) differentiated from cystic fibrosis (CF) iPSCs recapitulate the primary defects conferred by different types of CF mutations. LPCs enable high-throughput testing of interventions, and pilot studies show that responses in LPCs can be validated in patient-matched primary nasal epithelial cultures, confirming the potential utility of LPCs in precision CF therapy development.
Pulmonary disease is the major cause of morbidity and mortality in patients with cystic fibrosis, a disease caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. ...Heterogeneity in CFTR genotype-phenotype relationships in affected individuals plus the escalation of drug discovery targeting specific mutations highlights the need to develop robust in vitro platforms with which to stratify therapeutic options using relevant tissue. Toward this goal, we adapted a fluorescence plate reader assay of apical CFTR-mediated chloride conductance to enable profiling of a panel of modulators on primary nasal epithelial cultures derived from patients bearing different CFTR mutations. This platform faithfully recapitulated patient-specific responses previously observed in the "gold-standard" but relatively low-throughput Ussing chamber. Moreover, using this approach, we identified a novel strategy with which to augment the response to an approved drug in specific patients. In proof of concept studies, we also validated the use of this platform in measuring drug responses in lung cultures differentiated from cystic fibrosis iPS cells. Taken together, we show that this medium throughput assay of CFTR activity has the potential to stratify cystic fibrosis patient-specific responses to approved drugs and investigational compounds in vitro in primary and iPS cell-derived airway cultures.
Induced Pluripotent Stem Cells (iPSCs) can be differentiated into epithelial organoids that recapitulate the relevant context for CFTR and enable testing of therapies targeting Cystic Fibrosis ...(CF)-causing mutant proteins. However, to date, CF-iPSC-derived organoids have only been used to study pharmacological modulation of mutant CFTR channel activity and not the activity of other disease-relevant membrane protein constituents. In the current work, we describe a high-throughput, fluorescence-based assay of CFTR channel activity in iPSC-derived intestinal organoids and describe how this method can be adapted to study other apical membrane proteins. Specifically, we show how this assay can be employed to study CFTR and ENaC channels and an electrogenic acid transporter in the same iPSC-derived intestinal tissue. This phenotypic platform promises to expand CF therapy discovery to include strategies that target multiple determinants of epithelial fluid transport.
Abstract only The sarco(endo)plasmic reticulum (SR/ER) is an essential regulator of many key cellular processes, especially those that play a role in the development and progression of cardiac ...disease. However, many aspects of its structural organization remain poorly defined. Receptor Expression Enhancing Protein 5 (REEP5) is a cardiac enriched SR/ER membrane protein, which regulates organization of the highly differentiated SR/ER network and responses to stress. In zebrafish models, genetic knock-out of reep5 results in cardiac functional defects and reduced heart rate. Within the cardiomyocyte, depletion of Reep5 in vitro results in decreased muscle cell contraction, disrupted Ca 2+ signaling and SR/ER luminal vacuolization. For these studies, in vivo cardiac knock-down of Reep5 in the mouse was achieved using recombinant adeno-associated virus serotype 9 (rAAV9)-mediated gene delivery. Cardiac tissues or isolated cardiomyocytes were harvested at 7 days through to 4 weeks following knock-down, for biochemical and functional assessments. We observed that the largest significant change in REEP5 expression occurred 4 weeks post-rAAV9 injection, correlating to a 78% knock-down, observed by immunoblotting (unpaired t-test, p<0.0001, n=6-8). 5 weeks following knock-down, mice developed lethal cardiac dysfunction. To assess the biochemical changes induced by Reep5 knock-down, we have established an organelle-specific cardiac proteomic profile of the microsome, mitochondria and cytosol, using subcellular fractionation and mass spectrometry (nLC-ESI-MS-HCD-MS). Coupled with high resolution confocal microscopy and 3D mapping techniques, we have examined localization and expression patterns of key SR/ER and mitochondrial proteins, that have altered expression following knock-down of Reep5 at the myocyte-level. These findings provide a detailed understanding of the role that REEP5 plays in maintaining ER homeostasis, SR/ER structure, and general organelle integrity. By identifying the mechanistic significance of REEP5 expression in the heart, we can work to delineate underappreciated pathways in cardiac muscle development.
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