Global phosphoproteomic profiling reveals perturbed signaling in a mouse model of dilated cardiomyopathy

Phospholamban (PLN) plays a central role in Ca²⁺ homeostasis in cardiac myocytes through regulation of the sarco(endo)plasmic reticulum Ca²⁺-ATPase 2A (SERCA2A) Ca²⁺ pump. An inherited mutation converting arginine residue 9 in PLN to cysteine (R9C) results in dilated cardiomyopathy (DCM) in humans and transgenic mice, but the downstream signaling defects leading to decompensation and heart failure are poorly understood. Here we used precision mass spectrometry to study the global phosphorylation dynamics of 1,887 cardiac phosphoproteins in early affected heart tissue in a transgenic R9C mouse model of DCM compared with wild-type littermates. Dysregulated phosphorylation sites were quantified after affinity capture and identification of 3,908 phosphopeptides from fractionated whole-heart homogenates. Global statistical enrichment analysis of the differential phosphoprotein patterns revealed selective perturbation of signaling pathways regulating cardiovascular activity in early stages of DCM. Strikingly, dysregulated signaling through the Notch-1 receptor, recently linked to cardiomyogenesis and embryonic cardiac stem cell development and differentiation but never directly implicated in DCM before, was a prominently perturbed pathway. We verified alterations in Notch-1 downstream components in early symptomatic R9C transgenic mouse cardiomyocytes compared with wild type by immunoblot analysis and confocal immunofluorescence microscopy. These data reveal unexpected connections between stress-regulated cell signaling networks, specific protein kinases, and downstream effectors essential for proper cardiac function.Cardiovascular diseases (CVDs) leading to systolic/diastolic heart failure (HF), such as hypertensive/diabetic heart disease, stroke, and vascular atherosclerosis, are leading causes of death in the developed world (1). Many CVDs are associated with genetic predispositions. For example, in humans, the arginine to cysteine (R9C) substitution in phospholamban (PLN) has been shown to result in dilated cardiomyopathy (DCM) presenting in adolescence, leading to rapid deterioration of heart function and premature death (2). However, the etiology and molecular mechanisms of progression of DCM and other CVDs leading to HF are complex and still poorly understood, further complicating clinical assessment and management. From a biological and clinical perspective, the identification and characterization of clinically relevant, potentially druggable, pathways driving the maladaptive response in affected heart tissue are key challenges to improved diagnostic and therapeutic tools for earlier detection and preventative treatment of both inherited and chronic CVDs.Cardiac muscle contraction is controlled by Ca²⁺ flux and signaling relays, which are perturbed in HF. Internal stores of Ca²⁺ required for the proper functioning of cardiomyocytes (CMs) are normally maintained through the function of the sarco(endo)plasmic reticulum Ca²⁺-ATPase 2 (SERCA2) (3), which is responsible for the sequestration of Ca²⁺ resulting in muscle relaxation. SERCA2 activity is regulated through a reversible inhibitory interaction with PLN, which can be relieved by phosphorylation of PLN by protein kinase A (PKA) or Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) (3).Proteomic analyses have revealed changes in the abundance of other effector proteins in diverse biochemical pathways in DCM. Notably, shotgun proteomic analysis of membrane protein expression dynamics in heart microsomes isolated from mice overexpressing a superinhibitory (I40A) mutant of PLN revealed changes in G protein-coupled receptor-mediated pathways leading to activation of protein kinase C (PKC) (4). We previously reported quantitative changes in protein and cognate mRNA expression levels in cardiac ventricular tissue at different time points in the development of DCM in R9C-PLN mice representing clear clinical stages in the progression to HF (5). We showed that the latter maladaptive response was driven by elevated activity of MAPK signaling by the protein kinases p38 and JNK, in part through down-regulation of prosurvival microRNAs (6). However, the underlying upstream and downstream signaling events preceding HF were not fully explored.In the present study, we report a systematic, large-scale quantitative phosphoproteomic analysis of dysregulated protein phosphorylation-dependent signaling occurring at the early symptomatic stages of DCM progression in whole hearts from R9C mutant mice compared with wild-type littermates.     

Attenuated expression of profilin-1 confers protection from atherosclerosis in the LDL receptor null mouse

Atherosclerosis-related events are a major cause of morbidity and death worldwide, but the mechanisms underlying atherogenesis are not fully understood. We showed in previous studies that the actin-binding protein profilin-1 (pfn) was upregulated in atherosclerotic plaques and in endothelial cells (ECs) treated with oxidized low-density lipoproteins (oxLDL). The present study addressed the role of pfn in atheroma formation. To this end, mice with heterozygous deficiency of pfn, Pfn(+/-), were crossed with Ldlr(-/-) mice. After 2 months under a 1.25% cholesterol atherogenic diet, Pfn(+/-)Ldlr(-/-) (PfnHet) exhibited a significant reduction in lesion burden compared with Ldlr(-/-) control mice (PfnWT), whereas total cholesterol and triglyceride levels were similar in the 2 groups. Relevant atheroprotective changes were identified in PfnHet. When compared with PfnWT, aortas from PfnHet mice showed preserved endothelial nitric oxide synthase (eNOS) activation and nitric oxide (NO)-dependent signaling, and reduced vascular cell adhesion molecule (VCAM)-1 expression and macrophage accumulation at lesion-prone sites. Similarly, knockdown of pfn in cultured aortic ECs was protective against endothelial dysfunction triggered by oxLDL. Finally, bone marrow-derived macrophages from PfnHet showed blunted internalization of oxLDL and oxLDL-induced inflammation. These studies demonstrate that pfn levels modulate processes critical for early atheroma formation and suggest that pfn heterozygosity confers atheroprotection through combined endothelial- and macrophage-dependent mechanisms.     

Gene expression profiling reveals stromal genes expressed in common between Barrett's esophagus and adenocarcinoma

BACKGROUND & AIMS: Barrett's esophagus is a precursor of esophageal adenocarcinoma. DNA microarrays that enable a genome-wide assessment of gene expression enhance the identification of specific genes as well as gene expression patterns that are expressed by Barrett's esophagus and adenocarcinoma compared with normal tissues. Barrett's esophagus length has also been identified as a risk factor for progression to adenocarcinoma, but whether there are intrinsic biological differences between short-segment and long-segment Barrett's esophagus can be explored with microarrays. METHODS: Gene expression profiles for endoscopically obtained biopsy specimens of Barrett's esophagus or esophageal adenocarcinoma and associated normal esophagus and duodenum were identified for 17 patients using DNA microarrays. Unsupervised and supervised approaches for data analysis defined similarities and differences between the tissues as well as correlations with clinical phenotypes. RESULTS: Each tissue displays a unique expression profile that distinguishes it from others. Barrett's esophagus and esophageal adenocarcinoma express a unique set of stromal genes that is distinct from normal tissues but similar to other cancers. Adenocarcinoma also showed lower and higher expression for many genes compared with Barrett's esophagus. No difference in gene expression was found between short-segment and long-segment Barrett's esophagus. CONCLUSIONS: The genome-wide assessment provided by current DNA microarrays reveals many candidate genes and patterns not previously identified. Stromal gene expression in Barrett's esophagus and adenocarcinoma is similar, indicating that these changes precede malignant transformation.     

Gene expression profiling identifies liver X receptor alpha as an estrogen-regulated gene in mouse adipose tissue

Estrogens reduce adipose tissue mass in both humans and animals. The molecular mechanisms for this effect are, however, not well characterized. We took a gene expression profiling approach to study the direct effects of estrogen on mouse white adipose tissue (WAT). Female ovariectomized mice were treated for 10, 24 and 48 h with 17beta-estradiol or vehicle. RNA was extracted from gonadal fat and hybridized to Affymetrix MG-U74Av2 arrays. 17beta-Estradiol was shown to decrease mRNA expression of liver X receptor (LXR) alpha after 10 h of treatment compared with the vehicle control. The expression of several LXRalpha target genes, such as sterol regulatory element-binding protein 1c, apolipoprotein E, phospholipid transfer protein, ATP-binding cassette A1 and ATP-binding cassette G1, was similarly decreased. We furthermore identified a 1.5 kb LXRalpha promoter fragment that is negatively regulated by estrogen. Several genes involved in lipogenesis and lipolysis were identified as novel targets that could mediate estrogenic effects on adipose tissue. Finally, we show that ERalpha is the main estrogen receptor expressed in mouse white adipose tissue (WAT) with mRNA levels several hundred times higher than those of ERbeta mRNA.     

Gene profiling of Chikungunya virus arthritis in a mouse model reveals significant overlap with rheumatoid arthritis

Objective

Chikungunya virus (CHIKV) is a mosquito‐borne alphavirus that causes a chronic debilitating polyarthralgia/polyarthritis, for which current treatments are often inadequate. To assess whether new drugs being developed for rheumatoid arthritis (RA) might find utility in the treatment of alphaviral arthritides, we sought to determine whether the inflammatory gene expression signature of CHIKV arthritis shows any similarities with RA or collagen‐induced arthritis (CIA), a mouse model of RA.

Methods

Using a recently developed animal model of CHIKV arthritis in adult wild‐type mice, we generated a consensus CHIKV arthritis gene expression signature, which was used to interrogate publicly available microarray studies of RA and CIA. Pathway analyses were then performed using the overlapping gene signatures.

Results

Gene set enrichment analysis showed that there was a highly significant overlap in the differentially expressed genes in the CHIKV arthritis model and in RA. This concordance also increased with the severity of RA, as measured by the inflammation score. A highly significant overlap was also seen between CHIKV arthritis and CIA. Pathway analysis revealed that the overlap between these arthritides was spread over a range of different inflammatory processes. Involvement of T cells and interferon‐γ (IFNγ) in CHIKV arthritis was confirmed in studies of MHCII‐deficient mice and IFNγ‐deficient mice, respectively.

Conclusion

These results suggest that RA, a chronic autoimmune arthritis, and CHIKV disease, usually a self‐limiting viral arthropathy, share multiple inflammatory processes. New drugs and biologic therapies being developed for RA may thus find application in the treatment of alphaviral arthritides.

     

Expression profiling of mouse subplate reveals a dynamic gene network and disease association with autism and schizophrenia

The subplate zone is a highly dynamic transient sector of the developing cerebral cortex that contains some of the earliest generated neurons and the first functional synapses of the cerebral cortex. Subplate cells have important functions in early establishment and maturation of thalamocortical connections, as well as in the development of inhibitory cortical circuits in sensory areas. So far no role has been identified for cells in the subplate in the mature brain and disease association of the subplate-specific genes has not been analyzed systematically. Here we present gene expression evidence for distinct roles of the mouse subplate across development as well as unique molecular markers to extend the repertoire of subplate labels. Performing systematic comparisons between different ages (embryonic days 15 and 18, postnatal day 8, and adult), we reveal the dynamic and constant features of the markers labeling subplate cells during embryonic and early postnatal development and in the adult. This can be visualized using the online database of subplate gene expression at https://molnar.dpag.ox.ac.uk/subplate/. We also identify embryonic similarities in gene expression between the ventricular zones, intermediate zone, and subplate, and distinct postnatal similarities between subplate, layer 5, and layers 2/3. The genes expressed in a subplate-specific manner at some point during development show a statistically significant enrichment for association with autism spectrum disorders and schizophrenia. Our report emphasizes the importance of the study of transient features of the developing brain to better understand neurodevelopmental disorders.     

Global gene expression profiling in mouse plasma cell tumor precursor and bystander cells reveals potential intervention targets for plasma cell neoplasia

Tumor progression usually proceeds through several sequential stages, any of which could be targets for interrupting the progression process if one understood these steps at the molecular level. We extracted nascent plasma cell tumor (PCT) cells from within inflammatory oil granulomas (OG) isolated from IP pristane-injected BALB/c.iMyc(Eμ) mice at 5 different time points during tumor progression. We used laser capture microdissection to collect incipient PCT cells and analyzed their global gene expression on Affymetrix Mouse Genome 430A microarrays. Two independent studies were performed with different sets of mice. Analysis of the expression data used ANOVA and Bayesian estimation of temporal regulation. Genetic pathway analysis was performed using MetaCore (GeneGo) and IPA (Ingenuity). The gene expression profiles of PCT samples and those of undissected OG samples from adjacent sections showed that different genes and pathways were mobilized in the tumor cells during tumor progression, compared with their stroma. Our analysis implicated several genetic pathways in PCT progression, including biphasic (up- and then down-regulation) of the Spp1/osteopontin-dependent network and up-regulation of mRNA translation/protein synthesis. The latter led to a biologic validation study that showed that the AMPK-activating diabetes drug, metformin, was a potent specific PCT inhibitor in vitro.     

A novel type of familial hypercholesterolemia: double heterozygous mutations in LDL receptor and LDL receptor adaptor protein 1 gene

Background

Autosomal recessive hypercholesterolemia (ARH) is an extremely rare inherited hypercholesterolemia, the cause of which is mutations in low-density lipoprotein (LDL) receptor adaptor protein 1 (LDLRAP1) gene.

Methods

A total of 146 heterozygous familial hypercholesterolemic (FH) patients with a mutation in LDLR gene were screened for genes encoding proprotein convertase subtilisin/kexin type 9 (PCSK9) and LDLRAP1.

Results

Among the 146 subjects, we identified a 79-year-old Japanese female with double mutations in LDLR gene (c.2431A > T) and LDLRAP1 gene (c.606dup). Two other relatives with double mutations in those genes in her family were also identified. Although the proband exhibited massive Achilles tendon xanthoma and coronary and aortic valvular disease, serum LDL-C level of subjects with double mutations was similar with that of subjects with single LDLR mutation (284.0 ± 43.5 versus 265.1 ± 57.4 mg/dl).

Conclusion

Additional mutation in LDLRAP1 may account for severer phenotype in terms of xanthoma and atherosclerotic cardiovascular disease in FH patients.     

Increased LDL cholesterol and atherosclerosis in LDL receptor-deficient mice with attenuated expression of scavenger receptor B1

Scavenger receptor BI (SR-BI) is a multiligand cell-surface receptor that plays a central role in high density lipoprotein homeostasis in rodents. To investigate a role for SR-BI in atherosclerosis, mice with attenuated SR-BI expression were crossed with low density lipoprotein (LDL) receptor-deficient mice. Compound-homozygous mutants showed increased plasma cholesterol, surprisingly due primarily to increased LDL cholesterol and apolipoprotein B levels. LDL turnover studies showed that this resulted from increased LDL cholesterol production rather than decreased LDL catabolism. Atherosclerotic lesion size was significantly increased in male compound-mutant mice relative to LDL receptor-deficient controls (93 427+/-16 079 versus 34 448+/-5 331 microm(2), respectively; P=0.003). The proatherogenic effect of attenuated SR-BI expression may in part be due to increased LDL cholesterol levels. These findings suggest that upregulation of the receptor could have therapeutic potential for the treatment of atherosclerosis.     

Identification of novel epididymal genes by expression profiling and in silico gene discovery

We have used both the UniGene RIKEN epididymal EST library and the Affymetrix microarray profiling for identifying novel epididymal genes in mouse. The use of ESTs is a complementary approach to Affymetrix arrays for identifying novel epididymal genes, while only 32% and 28% of ESTs of unknown genes were present in the U74v.2Set and MG 430 2.0 version Affymetrix arrays, respectively. As expected, the probe set for a notably larger proportion of known genes was present in the Affymetrix arrays, and the coverage was greatly improved by the newer array version. Furthermore, many genes with more than five ESTs in the UniGene library showed variable levels of expression in both versions of the Affymetrix arrays. However, both the Affymetrix and EST data correlated well with that obtained by quantitative RT-PCR, and thus, we conclude that the findings of high EST number but only limited expression in the arrays could be considered as false negatives in the Affymetrix arrays.