Luca Brunelli

Peroxisome Proliferator-Activated Receptor-δ Upregulates 14-3-3ε in Human Endothelial Cells via CCAAT/Enhancer Binding Protein-β

Peroxisome proliferator-activated receptor &dgr; (PPAR&dgr;) agonists are promising new agents for treatment of the metabolic syndrome. Although they possess antiatherosclerotic properties in vivo and promote endothelial cell survival, their mechanism of action is incompletely understood. 14-3-3ϵ is a critical component of the endothelial cell antiapoptotic machinery, which is essential to maintain homeostasis of the vascular wall. To test the hypothesis that PPAR&dgr; targets 14-3-3ϵ in endothelial cells, we studied the response of the gene that encodes 14-3-3ϵ in humans, YWHAE, to PPAR&dgr; ligands (L-165,041 and GW501516). We found that PPAR&dgr; activates YWHAE promoter in a concentration and time-dependent manner. Consistent with these findings, L-165,041 increased 14-3-3ϵ mRNA and protein level, whereas PPAR&dgr; small interfering RNA suppressed both basal and L-165,041–dependent YWHAE transcription and 14-3-3ϵ protein expression. Surprisingly, PPAR response elements in YWHAE promoter were not required for upregulation by PPAR&dgr;, whereas a CCAAT/enhancer binding protein (C/EBP) site located at −160/−151 bp regulated both basal and PPAR&dgr;-dependent promoter activity. Intriguingly, activation or knock down of endogenous PPAR&dgr; regulated C/EBP&bgr; protein expression. Chromatin immunoprecipitation assays demonstrated that L-165,041 determines the localization of C/EBP&bgr; to the region spanning this C/EBP response element, whereas sequential chromatin immunoprecipitation analysis revealed that C/EBP&bgr; and PPAR&dgr; form a transcriptional activating complex on this C/EBP site. Our work uncovers a novel role for C/EBP&bgr; as a mediator of PPAR&dgr;-dependent 14-3-3ϵ gene regulation in human endothelial cells and provides insight into the mechanism by which PPAR&dgr; agonists may be beneficial in atherosclerosis.

A ZASP Missense Mutation, S196L, Leads to Cytoskeletal and Electrical Abnormalities in a Mouse Model of Cardiomyopathy

Background—Dilated cardiomyopathy (DCM) is a primary disease of the heart muscle associated with sudden cardiac death secondary to ventricular tachyarrhythmias and asystole. However, the molecular pathways linking DCM to arrhythmias and sudden cardiac death are unknown. We previously identified a S196L mutation in exon 4 of LBD3-encoded ZASP in a family with DCM and sudden cardiac death. These findings led us to hypothesize that this mutation may precipitate both cytoskeletal and conduction abnormalities in vivo. Therefore, we investigated the role of the ZASP4 mutation S196L in cardiac cytoarchitecture and ion channel biology. Methods and Results—We generated and analyzed transgenic mice with cardiac-restricted expression of the S196L mutation. We also performed cellular electrophysiological analysis on isolated S196L cardiomyocytes and protein-protein interaction studies. Ten month-old S196L mice developed hemodynamic dysfunction consistent with DCM, whereas 3-month-old S196L mice presented with cardiac conduction defects and atrioventricular block. Electrophysiological analysis on isolated S196L cardiomyocytes demonstrated that the L-type Ca2+ currents and Na+ currents were altered. The pull-down assay demonstrated that ZASP4 complexes with both calcium (Cav1.2) and sodium (Nav1.5) channels. Conclusions—Our findings provide new insight into the mechanisms by which mutations of a structural/cytoskeletal protein, such as ZASP, lead to cardiac functional and electric abnormalities. This work represents a novel framework to understand the development of conduction defects and arrhythmias in subjects with cardiomyopathies, including DCM.

14-3-3ε Plays a Role in Cardiac Ventricular Compaction by Regulating the Cardiomyocyte Cell Cycle

ABSTRACT Trabecular myocardium accounts for the majority of the ventricles during early cardiogenesis, but compact myocardium is the primary component at later developmental stages. Elucidation of the genes regulating compact myocardium development is essential to increase our understanding of left ventricular noncompaction (LVNC), a cardiomyopathy characterized by increased ratios of trabecular to compact myocardium. 14-3-3ε is an adapter protein expressed in the lateral plate mesoderm, but its in vivo cardiac functions remain to be defined. Here we show that 14-3-3ε is expressed in the developing mouse heart as well as in cardiomyocytes. 14-3-3ε deletion did not appear to induce compensation by other 14-3-3 isoforms but led to ventricular noncompaction, with features similar to LVNC, resulting from a selective reduction in compact myocardium thickness. Abnormal compaction derived from a 50% decrease in cardiac proliferation as a result of a reduced number of cardiomyocytes in G2/M and the accumulation of cardiomyocytes in the G0/G1 phase of the cell cycle. These defects originated from downregulation of cyclin E1 and upregulation of p27Kip1, possibly through both transcriptional and posttranslational mechanisms. Our work shows that 14-3-3ε regulates cardiogenesis and growth of the compact ventricular myocardium by modulating the cardiomyocyte cell cycle via both cyclin E1 and p27Kip1. These data are consistent with the long-held view that human LVNC may result from compaction arrest, and they implicate 14-3-3ε as a new candidate gene in congenital human cardiomyopathies.

A Family-Based Paradigm to Identify Candidate Chromosomal Regions for Isolated Congenital Diaphragmatic Hernia

Congenital diaphragmatic hernia (CDH) is a developmental defect of the diaphragm that causes high newborn mortality. Isolated or non‐syndromic CDH is considered a multifactorial disease, with strong evidence implicating genetic factors. As low heritability has been reported in isolated CDH, family‐based genetic methods have yet to identify the genetic factors associated with the defect. Using the Utah Population Database, we identified distantly related patients from several extended families with a high incidence of isolated CDH. Using high‐density genotyping, seven patients were analyzed by homozygosity exclusion rare allele mapping (HERAM) and phased haplotype sharing (HapShare), two methods we developed to map shared chromosome regions. Our patient cohort shared three regions not previously associated with CDH, that is, 2q11.2–q12.1, 4p13 and 7q11.2, and two regions previously involved in CDH, that is, 8p23.1 and 15q26.2. The latter regions contain GATA4 and NR2F2, two genes implicated in diaphragm formation in mice. Interestingly, three patients shared the 8p23.1 locus and one of them also harbored the 15q26.2 segment. No coding variants were identified in GATA4 or NR2F2, but a rare shared variant was found in intron 1 of GATA4. This work shows the role of heritability in isolated CDH. Our family‐based strategy uncovers new chromosomal regions possibly associated with disease, and suggests that non‐coding variants of GATA4 and NR2F2 may contribute to the development of isolated CDH. This approach could speed up the discovery of the genes and regulatory elements causing multifactorial diseases, such as isolated CDH.

Loss of Function of hNav1.5 by a ZASP1 Mutation Associated With Intraventricular Conduction Disturbances in Left Ventricular Noncompaction

Background—Defects of cytoarchitectural proteins can cause left ventricular noncompaction, which is often associated with conduction system diseases. We have previously identified a p.D117N mutation in the LIM domain-binding protein 3–encoding Z-band alternatively spliced PDZ motif gene (ZASP) in a patient with left ventricular noncompaction and conduction disturbances. We sought to investigate the role of p.D117N mutation in the LBD3 NM_001080114.1 isoform (ZASP1-D117N) for the regulation of cardiac sodium channel (Nav1.5) that plays an important role in the cardiac conduction system. Methods and Results—Effects of ZASP1-wild-type and ZASP1-D117N on Nav1.5 were studied in human embryonic kidney-293 cells and neonatal rat cardiomyocytes. Patch-clamp study demonstrated that ZASP1-D117N significantly attenuated INa by 27% in human embryonic kidney-293 cells and by 32% in neonatal rat cardiomyocytes. In addition, ZASP1-D117N rightward shifted the voltage-dependent activation and inactivation in both systems. In silico simulation using Luo-Rudy phase 1 model demonstrated that altered Nav1.5 function can reduce cardiac conduction velocity by 28% compared with control. Pull-down assays showed that both wild-type and ZASP1-D117N can complex with Nav1.5 and telethonin/T-Cap, which required intact PDZ domains. Immunohistochemical staining in neonatal rat cardiomyocytes demonstrates that ZASP1-D117N did not significantly disturb the Z-line structure. Disruption of cytoskeletal networks with 5-iodonaphthalene-1-sulfonyl homopiperazine and cytochalasin D abolished the effects of ZASP1-D117N on Nav1.5. Conclusions—ZASP1 can form protein complex with telethonin/T-Cap and Nav1.5. The left ventricular noncompaction-specific ZASP1 mutation can cause loss of function of Nav1.5, without significant alteration of the cytoskeletal protein complex. Our study suggests that electric remodeling can occur in left ventricular noncompaction subject because of a direct effect of mutant ZASP on Nav1.5.

14-3-3ε Gene variants in a Japanese patient with left ventricular noncompaction and hypoplasia of the corpus callosum

BACKGROUND Left ventricular noncompaction (LVNC) is a cardiomyopathy characterized by a prominent trabecular meshwork and deep intertrabecular recesses, and is thought to be due to an arrest of normal endomyocardial morphogenesis. However, the genes contributing to this process remain poorly understood. 14-3-3ε, encoded by YWHAE, is an adapter protein belonging to the 14-3-3 protein family which plays important roles in neuronal development and is involved in Miller-Dieker syndrome. We recently showed that mice lacking this gene develop LVNC. Therefore, we hypothesized that variants in YWHAE may contribute to the pathophysiology of LVNC in humans. METHODS AND RESULTS In 77 Japanese patients with LVNC, including the probands of 29 families, mutation analysis of YWHAE by direct DNA sequencing identified 7 novel variants. One of them, c.-458G>T, in the YWHAE promoter, was identified in a familial patient with LVNC and hypoplasia of the corpus callosum. The -458G>T variant is located within a regulatory CCAAT/enhancer binding protein (C/EBP) response element of the YWHAE promoter, and it reduced promoter activity by approximately 50%. Increased binding of an inhibitory C/EBPβ isoform was implicated in decreasing YWHAE promoter activity. Interestingly, we had previously shown that C/EBPβ is a key regulator of YWHAE. CONCLUSIONS These data suggest that the -458G>T YWHAE variant contributes to the abnormal myocardial morphogenesis characteristic of LVNC as well as abnormal brain development, and implicate YWHAE as a novel candidate gene in pediatric cardiomyopathies.

A One-Step Miniprep for the Isolation of Plasmid DNA and Lambda Phage Particles

Plasmid DNA minipreps are fundamental techniques in molecular biology. Current plasmid DNA minipreps use alkali and the anionic detergent SDS in a three-solution format. In addition, alkali minipreps usually require additional column-based purification steps and cannot isolate other extra-chromosomal elements, such as bacteriophages. Non-ionic detergents (NIDs) have been used occasionally as components of multiple-solution plasmid DNA minipreps, but a one-step approach has not been developed. Here, we have established a one-tube, one-solution NID plasmid DNA miniprep, and we show that this approach also isolates bacteriophage lambda particles. NID minipreps are more time-efficient than alkali minipreps, and NID plasmid DNA performs better than alkali DNA in many downstream applications. In fact, NID crude lysate DNA is sufficiently pure to be used in digestion and sequencing reactions. Microscopic analysis showed that the NID procedure fragments E.coli cells into small protoplast-like components, which may, at least in part, explain the effectiveness of this approach. This work demonstrates that one-step NID minipreps are a robust method to generate high quality plasmid DNA, and NID approaches can also isolate bacteriophage lambda particles, outperforming current standard alkali-based minipreps.

Hofmeister series salts enhance purification of plasmid DNA by non-ionic detergents

Ion‐exchange chromatography is the standard technique used for plasmid DNA purification, an essential molecular biology procedure. Non‐ionic detergents (NIDs) have been used for plasmid DNA purification, but it is unclear whether Hofmeister series salts (HSS) change the solubility and phase separation properties of specific NIDs, enhancing plasmid DNA purification. After scaling‐up NID‐mediated plasmid DNA isolation, we established that NIDs in HSS solutions minimize plasmid DNA contamination with protein. In addition, large‐scale NID/HSS solutions eliminated lipopolysaccharides (LPS) contamination of plasmid DNA more effectively than Qiagen ion‐exchange columns. Large‐scale NID isolation/NID purification generated increased yields of high‐quality DNA compared to alkali isolation/column purification. This work characterizes how HSS enhance NID‐mediated plasmid DNA purification, and demonstrates that NID phase transition is not necessary for LPS removal from plasmid DNA. Specific NIDs such as IGEPAL CA‐520 can be utilized for rapid, inexpensive, and efficient laboratory‐based large‐scale plasmid DNA purification, outperforming Qiagen‐based column procedures. Biotechnol. Bioeng. 2011; 108:1872–1882.

Remodeling of dystrophin and sarcomeric Z-band occurs in pediatric cardiomyopathies: a unifying mechanism for force transmission defect.

Background Cardiomyopathies (CMPs) lead to associated systolic dysfunction and are the major causes of congestive heart failure and a leading cause for heart transplantation. Although the precise mechanism leading to systolic dysfunction is still elusive, chronic mechanical loading, along with altered calcium (Ca2+) cellular homeostasis, is believed to impair force transmission and induce cardiac morphological and structural changes, namely cardiac remodeling. Interestingly, dystrophin remodeling has been previously reported to occur in adults with end-stage CMP irrespective of the underlying cause. Methods In order to determine the structural culprit associated with pediatric dilated cardiomyopathy (DCM) due to various causes, we investigated the structural continuum connecting dystrophin and the dystrophin-associated glycoprotein complex to the contractile apparatus in heart samples from four children with idiopathic dilated CMP: one with myocarditis, one sporadic DCM child previously identified with a δ-sarcoglycan deletion mutation, and one child with X-linked CMP with a reported splicing site mutation in the dystrophin-coded DYS gene. Results Immunohistochemical analysis of cytoskeletal proteins connecting the dystrophin-associated glycoprotein complex to the sarcomere identified that myocarditis, idiopathic, and genetic-based DCM are characterized by disruption of the dystrophin connection to the sarcomere and perturbation of the Z-band. Conclusion Our data suggest that both dystrophin remodeling and sarcomeric Z-band/disk derangements may occur in the myocardium of children with DCM irrespective of the cause. This suggests that genetic mutations in the dystrophin-associated glycoprotein complex or any of its partners could result in sarcomere–sarcolemma connection alteration and associated Z-band disturbance, thus leading to force transmission dysfunction.

Isolation of rare recombinants without using selectable markers for one-step seamless BAC mutagenesis

Current methods to isolate rare (1:10,000–1:100,000) bacterial artificial chromosome (BAC) recombinants require selectable markers. For seamless BAC mutagenesis, selectable markers need to be removed after isolation of recombinants through counterselection. Here we illustrate founder principle–driven enrichment (FPE), a simple method to rapidly isolate rare recombinants without using selectable markers, allowing one-step seamless BAC mutagenesis. As proof of principle, we isolated 1:100,000 seamless fluorescent protein–modified Nodal BACs and confirmed BAC functionality by generating fluorescent reporter mice. We also isolated small indel P1 phage–derived artificial chromosome (PAC) and BAC recombinants. Statistical analysis revealed that 1:100,000 recombinants can be isolated with <40 PCRs, and we developed a web-based calculator to optimize FPE.