Although it is generally recognized that cystic fibrosis transmembrane conductance regulator (CFTR) contains a PSD-95/Disc-large/ZO-1 (PDZ)-binding motif at its COOH terminus, the identity of the PDZ domain protein(s) that interact with CFTR is uncertain, and the functional impact of this interaction is not fully understood. By using human airway epithelial cells, we show that CFTR associates with Na ؉ /H ؉ exchanger (NHE) type 3 kinase A regulatory protein (E3KARP), an EBP50/ NHE regulatory factor (NHERF)-related PDZ domain protein. The PDZ binding motif located at the COOH terminus of CFTR interacts preferentially with the second PDZ domain of E3KARP, with nanomolar affinity. In contrast to EBP50/NHERF, E3KARP is predominantly localized (>95%) in the membrane fractions of Calu-3 and T84 cells, where CFTR is located. Moreover, confocal immunofluorescence microscopy of polarized Calu-3 monolayers shows that E3KARP and CFTR are co-localized at the apical membrane domain. We also found that ezrin associates with E3KARP in vivo. Co-expression of CFTR with E3KARP and ezrin in Xenopus oocytes potentiated cAMP-stimulated CFTR Cl ؊ currents. These results support the concept that E3KARP functions as a scaffold protein that links CFTR to ezrin. Since ezrin has been shown previously to function as a protein kinase A anchoring protein, we suggest that one function served by the interaction of E3KARP with both ezrin and CFTR is to localize protein kinase A in the vicinity of the R-domain of CFTR. Since ezrin is also an actinbinding protein, the formation of a CFTR⅐E3KARP⅐ezrin complex may be important also in stabilizing CFTR at the apical membrane domain of airway cells.

One million patients with congenital heart disease (CHD) live in the United States. They have a lifelong risk of developing heart failure. Current concepts do not sufficiently address mechanisms of heart failure development specifically for these patients. Here, analysis of heart tissue from an infant with tetralogy of Fallot with pulmonary stenosis (ToF/PS) labeled with isotope-tagged thymidine demonstrated that cardiomyocyte cytokinesis failure is increased in this common form of CHD. We used single-cell transcriptional profiling to discover that the underlying mechanism of cytokinesis failure is repression of the cytokinesis gene ECT2, downstream of β-adrenergic receptors (β-ARs). Inactivation of the β-AR genes and administration of the β-blocker propranolol increased cardiomyocyte division in neonatal mice, which increased the number of cardiomyocytes (endowment) and conferred benefit after myocardial infarction in adults. Propranolol enabled the division of ToF/PS cardiomyocytes in vitro. These results suggest that β-blockers could be evaluated for increasing cardiomyocyte division in patients with ToF/PS and other types of CHD.

The voltage-gated cardiac Na+ channel (Nav1.5), encoded by the SCN5A gene, conducts the inward depolarizing cardiac Na+ current (INa) and is vital for normal cardiac electrical activity. Inherited loss-of-function mutations in SCN5A lead to defects in the generation and conduction of the cardiac electrical impulse and are associated with various arrhythmia phenotypes1. Here we show that sirtuin 1 deacetylase (Sirt1) deacetylates Nav1.5 at lysine 1479 (K1479) and stimulates INa via lysine-deacetylation-mediated trafficking of Nav1.5 to the plasma membrane. Cardiac Sirt1 deficiency in mice induces hyperacetylation of K1479 in Nav1.5, decreases expression of Nav1.5 on the cardiomyocyte membrane, reduces INa and leads to cardiac conduction abnormalities and premature death owing to arrhythmia. The arrhythmic phenotype of cardiac-Sirt1-deficient mice recapitulated human cardiac arrhythmias resulting from loss of function of Nav1.5. Increased Sirt1 activity or expression results in decreased lysine acetylation of Nav1.5, which promotes the trafficking of Nav1.5 to the plasma membrane and stimulation of INa. As compared to wild-type Nav1.5, Nav1.5 with K1479 mutated to a nonacetylatable residue increases peak INa and is not regulated by Sirt1, whereas Nav1.5 with K1479 mutated to mimic acetylation decreases INa. Nav1.5 is hyperacetylated on K1479 in the hearts of patients with cardiomyopathy and clinical conduction disease. Thus, Sirt1, by deacetylating Nav1.5, plays an essential part in the regulation of INa and cardiac electrical activity.

Objective-Low-density lipoprotein (LDL) cholesterol induces endothelial dysfunction and is a major modifiable risk factor for coronary heart disease. Endothelial Kruppel-like Factor 2 (KLF2) is a transcription factor that is vital to endotheliumdependent vascular homeostasis. The purpose of this study is to determine whether and how LDL affects endothelial KLF2 expression. Approach and Results-LDL downregulates KLF2 expression and promoter activity in endothelial cells. LDL-induced decrease in KLF2 parallels changes in endothelial KLF2 target genes thrombomodulin, endothelial NO synthase, and plasminogen activator inhibitor-1. Pharmacological inhibition of DNA methyltransferases or knockdown of DNA methyltransferase 1 prevents downregulation of endothelial KLF2 by LDL. LDL induces endothelial DNA methyltransferase 1 expression and DNA methyltransferase activity. LDL stimulates binding of the DNA methylCpG-binding protein-2 and histone methyltransferase enhancer of zeste homolog 2, whereas decreases binding of the KLF2 transcriptional activator myocyte enhancing factor-2, to the KLF2 promoter in endothelial cells. Knockdown of myocyte enhancing factor-2, or mutation of the myocyte enhancing factor-2 site in the KLF2 promoter, abrogates LDLinduced downregulation of endothelial KLF2 and thrombomodulin, and KLF2 promoter activity. Similarly, knockdown of enhancer of zeste homolog 2 negates LDL-induced downregulation of KLF2 and thrombomodulin in endothelial cells. for myocyte enhancing factor-2 (MEF2), a transcription factor which upregulates KLF2, 9,10,15 and the tumor suppressing transcription factor p53 which downregulates KLF2. 16 Although studies on epigenetic modulation of KLF2 expression are limited, emerging evidence suggests that histone modifiers, such as the histone methyltransferase enhancer of zeste homolog 2 (EZH2), the catalytic subunit of Polycomb repressive complex 2 which is implicated in tumorigenesis and is responsible for trimethylation of Histone 3 on lysine 27, play an important part in governing KLF2 expression. 17Cholesterol-rich lipid particles and hypercholesterolemia are associated with epigenetic changes in vitro and in experimental animal models as well as in humans. ApoE −/− mice fed a high-fat diet have aberrant DNA methylation patterns, including decreased global methylation in aorta and peripheral blood mononuclear cells, 18 and human atherosclerotic tissue samples display genomic hypomethylation.19 P66shc is one of the genes epigenetically modified by cholesterolrich proatherogenic particles, such as LDL.20 P66shc mediates endothelial oxidative stress and fatty streak formation in hypercholesterolemic mice. 21 However, p66shc is only one of many genes whose epigenetic modification by hypercholesterolemia may contribute to the development of vascular disease. Intrigued by the possibility that endothelial KLF2 might also be epigenetically regulated in a hypercholesterolemic environment, we asked whether LDL cholesterol changes endothelial KLF2 transcription, and investigated the rol...

In support of this idea, ⌬NEG2 CFTR escaped from the inhibition of wild type CFTR trafficking produced by overexpression of syntaxin 1A. These observations suggest that the NEG2 region at the C terminus of the R domain allows stabilization of CFTR in a regulated intracellular compartment from which it traffics to the plasma membrane in response to cAMP/PKA stimulation. The cystic fibrosis transmembrane conductance regulator (CFTR)2 is a phosphorylation-activated anion channel located at the apical membranes of airway, intestinal, pancreatic, and salivaryglandepithelialcells.Itsstimulation,primarilybycAMPdependent signaling pathways, is the basis of electrolyte and fluid secretion that provides the fluid vehicle for macromolecular secretory products. In airway epithelia, CFTR is the principal apical anion conductance contributing to chloride and HCO 3 secretion, and this establishes the electrical and osmotic driving forces for secondary sodium and water transport. Together, these events regulate the volume and composition of the airway surface liquid (3, 4). Mutations in the gene encoding CFTR cause cystic fibrosis by either reducing its apical membrane density or interfering with its ability to transport anions (4). Identification of the primary amino acid sequence (5) placed CFTR in the ATP-binding cassette (ABC) transporter superfamily, of which there are ϳ50 members in the human genome. Similar to other ABC transporters (6), the N terminus of CFTR leads to six membrane-spanning segments that comprise the first transmembrane domain (TMD1), followed by a nucleotide-binding domain (NBD1). These structural elements are repeated in the C-terminal half of CFTR, as TMD2 and NBD2, followed by a C-terminal tail. A unique feature of CFTR among ABC family members is the presence of a regulatory (R) domain, interposed between these repeated TMD-NBD elements, whose multiple phosphorylation sites mediate cAMPdependent channel activation by protein kinase A (PKA) (7). The multiple PKA phosphorylation sites of the R domain act in concert to enable gating, and site mutagenesis has not revealed a requirement for phosphorylation at specific loci (8). Rather, there is redundancy in the ability of R domain PKA sites to support channel activation. The unstructured nature of the R domain (9) was confirmed in recent NMR structural studies, which showed that this largely disordered region contains segments of helical structure that likely interact with other CFTR domains, and perhaps other proteins, to effect its regulatory functions (10).Phosphorylation of the R domain is required for channel gating, which is then driven by the binding and hydrolysis of ATP at the NBDs of CFTR (11)(12)(13)(14). The formation of a head-to-tail NBD1/NBD2 dimer is thought to create shared ATP-binding sites that are contributed by residues from both NBDs, an arrangement based on bacterial ABC transporter structures (15). Conversely, the reversal of PKA-mediated channel activation requires R domain dephosphorylation, which is facilitated by phosphatases...

ABSTRACT/SUMMARYOne million patients with congenital heart disease (CHD) live in the US. They have a lifelong risk of developing heart failure. Current concepts do not sufficiently address mechanisms of heart failure development specifically for these patients. We show that cardiomyocyte cytokinesis failure is increased in tetralogy of Fallot with pulmonary stenosis (ToF/PS), a common form of CHD. Labeling of a ToF/PS baby with isotope-tagged thymidine showed cytokinesis failure after birth. We used single-cell transcriptional profiling to discover that the underlying mechanism is repression of the cytokinesis gene ECT2, and show that this is downstream of β-adrenergic receptors (β-AR). Inactivation of the β-AR genes and administration of the β-blocker propranolol increased cardiomyocyte division in neonatal mice, which increased the endowment and conferred benefit after myocardial infarction in adults. Propranolol enabled the division of ToF/PS cardiomyocytes. These results suggest that β-blockers should be evaluated for increasing cardiomyocyte division in patients with ToF/PS and other types of CHD.

Physiological calcium (Ca(2+)) signals within the pancreatic acinar cell regulate enzyme secretion, whereas aberrant Ca(2+) signals are associated with acinar cell injury. We have previously identified the ryanodine receptor (RyR), a Ca(2+) release channel on the endoplasmic reticulum, as a modulator of these pathological signals. In the present study, we establish that the RyR is expressed in human acinar cells and mediates acinar cell injury. We obtained pancreatic tissue from cadaveric donors and identified isoforms of RyR1 and RyR2 by qPCR. Immunofluorescence staining of the pancreas showed that the RyR is localized to the basal region of the acinar cell. Furthermore, the presence of RyR was confirmed from isolated human acinar cells by tritiated ryanodine binding. To determine whether the RyR is functionally active, mouse or human acinar cells were loaded with the high-affinity Ca(2+) dye (Fluo-4 AM) and stimulated with taurolithocholic acid 3-sulfate (TLCS) (500 μM) or carbachol (1 mM). Ryanodine (100 μM) pretreatment reduced the magnitude of the Ca(2+) signal and the area under the curve. To determine the effect of RyR blockade on injury, human acinar cells were stimulated with pathological stimuli, the bile acid TLCS (500 μM) or the muscarinic agonist carbachol (1 mM) in the presence or absence of the RyR inhibitor ryanodine. Ryanodine (100 μM) caused an 81% and 47% reduction in acinar cell injury, respectively, as measured by lactate dehydrogenase leakage (P < 0.05). Taken together, these data establish that the RyR is expressed in human acinar cells and that it modulates acinar Ca(2+) signals and cell injury.