bluuebunny/crossref_metadata_2025 huggingface dataset:
Implications of ventricular arrhythmia vulnerability during murine electrophysiology studies
Authors: Maguire Colin T. , Wakimoto Hiroko , Patel Vickas V. , Hammer Peter E. , Gauvreau Kimberlee , Berul Charles I.
Published: March 2015

Programmed ventricular stimulation is being performed for the provocation of ventricular arrhythmias in genetically engineered mice. Despite the high level of interest in this area of translational research, little attention has been given to differentiating between selectivity and specificity of induced ventricular tachycardia (VT) in phenotypically normal mice. We aimed to assess factors that may enhance inducibility of VT in wild-type (WT) mice. In vivo intracardiac electrophysiological studies (EPS) were performed in 230 WT mice of 4 strains. An octapolar electrode catheter was inserted into a jugular vein and advanced to the right atrium and ventricle. Baseline ventricular conduction, refractoriness, and arrhythmia inducibility were assessed using programmed electrical stimulation (PES) and burst pacing. We found that nonsustained VT (≥4 beats) was inducible in 68/230 (30%) mice. Duration of VT was 1.6 ± 2.4 s, and the longest episode lasted 24 s. VT inducibility differed by strain and age. Ventricular effective refractory period (VERP) was shorter in mice with inducible VT (44 ± 12 ms) compared with noninducible mice (61 ± 16 ms, P < 0.001). VERP increased with age ( P < 0.001), albeit with strain-related variability. We conclude that nonsustained VT in WT mice is reproducibly inducible and common. Genetic background variability may predispose certain strains to a higher incidence of arrhythmia induction. EPS methods impact prevalence and specificity of inducible VT. Increased VT inducibility was seen with shorter coupling intervals and application of tightly coupled extrastimuli techniques. These factors should be carefully considered when analyzing PES and burst pacing data in murine models to minimize false positives and optimize accuracy.
Novel technique of aortic banding followed by gene transfer during hypertrophy and heart failure
Authors: Del Monte Federica , Butler Karyn , Boecker Wolfgang , Gwathmey Judith K. , Hajjar Roger J.
Published: March 2015

Aortic banding in the rat has become a popular method to induce left ventricular (LV) hypertrophy and heart failure. However, because of often extensive intrathoracic adhesions and inflammatory cell infiltrates resulting from the traditional surgical approach, an uncomplicated second thoracic incision for genetic manipulation is impeded. In this study, we describe a novel surgical technique of aortic banding which avoids opening the sternum and thereby avoids adhesions and surgery-related inflammation. Placing a clip on the ascending aorta using a suprasternal approach in Sprague-Dawley rats created proximal aortic constriction. The present study was initiated to determine whether a replication-deficient adenovirus would enable efficient gene transfer to adult cardiac myocytes undergoing hypertrophy and transitioning to heart failure. Echocardiography performed at week 24 revealed significant concentric hypertrophy and increased fractional shortening followed by LV dilatation with decreased fractional shortening after 27 wk of banding. An adenoviral solution encoding for the reporter green fluorescent protein gene (GFP) was delivered to the heart. Fluorescent microscopy revealed global gene expression throughout hypertrophied and failing hearts. Our studies demonstrate that a novel suprasternal approach can be applied to create an LV hypertrophy model followed by heart failure which also allows investigators to perform genetic manipulations in vivo through gene transfer without the complication of adhesions and surgical trauma-induced inflammation. Furthermore, our approach to delivery of transgenes results in homogenous gene expression in both hypertrophied and failing hearts.
Differential regulation of gene expression by ovariectomy in mouse aorta
Authors: Villablanca Amparo C. , Lewis Kristine A. , Tham Doris , Rutledge John C.
Published: March 2015

The purpose of this study was to investigate the effects of ovarian hormones on gene expression in the vascular wall. Our approach employed an RT-PCR-based cloning strategy of DNA differential display analysis and verification/confirmation of differential expression by semi-quantitative PCR and real-time PCR. mRNA analysis of normal aortas from intact and ovariectomized female C57BL/6J mice, showed altered expression of 20 genes with significant (>70%) sequence homology to known genes. Eight were selected for further study based on the genes’ known function and potential relevance to vascular physiology. Differential expression of mRNA for three genes was confirmed by both semi-quantitative and real-time RT-PCR using gene-specific primers. Ovariectomy downregulated expression of elongation factor-1α (3.5-fold), ganglioside-induced differentiation associated protein (8.2-fold), and NADH:ubiquinone oxidoreductase (3.8-fold). Thus, in normal mouse aortas, ovariectomy resulted in significant differential downregulation of a number of vascular genes important to vascular cell growth and angiogenesis, cellular differentiation, and mitochondrial energy metabolism, respectively. These studies have implications for our understanding of hormonal regulation of vascular gene expression and the therapeutic targeting of specific vascular genetic sequences by female sex steroid hormones.
Transcriptional profiling identifies extensive downregulation of extracellular matrix gene expression in sarcopenic rat soleus muscle
Authors: Pattison J. Scott , Folk Lillian C. , Madsen Richard W. , Childs Thomas E. , Booth Frank W.
Published: March 2015

The direction of change in skeletal muscle mass differs between young and old individuals, growing in young animals and atrophying in old animals. The purpose of the experiment was to develop a statistically conservative list of genes whose expression differed significantly between young growing and old atrophying (sarcopenic) skeletal muscles, which may be contributing to physical frailty. Gene expression levels of >24,000 transcripts were determined in soleus muscle samples from young (3–4 mo) and old (30–31 mo) rats. Age-related differences were determined using a Student’s t-test (α of 0.05) with a Bonferroni adjustment, which yielded 682 probe sets that differed significantly between young ( n = 25) and old ( n = 20) animals. Of 347 total decreases in aged/sarcopenic muscle relative to young muscles, 199 were functionally identified; the major theme being that 24% had a biological role in the extracellular matrix and cell adhesion. Three themes were observed from 213 of the 335 total increases in sarcopenic muscles whose functions were documented in databases: 1) 14% are involved in immune response; 2) 9% play a role in proteolysis, ubiquitin-dependent degradation, and proteasome components; and 3) 7% act in stress/antioxidant responses. A total of 270 differentially expressed genes and ESTs had unknown/unclear functions. By decreasing the sample sizes of young and old animals from 25 × 20 to 15 × 15, 10 × 10, and 5 × 5 observations, we observed 682, 331, 73, and 3 statistically different mRNAs, respectively. Use of large sample size and a Bonferroni multiple testing adjustment in combination yielded increased statistical power, while protecting against false positives. Finally, multiple mRNAs that differ between young growing and old, sarcopenic muscles were identified and may highlight new candidate mechanisms that regulate skeletal muscle mass during sarcopenia.
Identification of AKT-regulated genes in inducible MERAkt cells
Authors: KUHN IRENE , BARTHOLDI MARTY F. , SALAMON HUGH , FELDMAN RICHARD I. , ROTH RICHARD A. , JOHNSON PAUL H.
Published: March 2015

AKT/protein kinase B plays a critical role in the phosphoinositide 3-kinase (PI3-kinase) pathway regulating cell growth, differentiation, and oncogenic transformation. Akt1-regulated genes were identified by cDNA array hybridization analysis using an inducible AKT1 protein, MERAKT. Treatment of MERAkt cells with estrogen receptor ligands resulted in phosphorylative activation of MERAKT. Genes differentially expressed in MERAkt/NIH3T3 cells treated with tamoxifen, raloxifene, ICI-182780, and ZK955, were identified at 3 and 20 h. AKT activation resulted in the repression of c-myc, early growth response 1 (EGR1), transforming growth factor β receptor III (TGF-βr III), and thrombospondin-1 (THBS1). Although c-myc induction is often associated with oncogenic transformation, the c-myc repression observed here is consistent with the anti-apoptotic function of AKT. Repression of THBS1 and EGR1 is consistent with the known pro-angiogenic functions of AKT. AKT-regulated genes were found to be largely distinct from platelet-derived growth factor-β (PDGFβ)-regulated genes; only T-cell death-associated gene 51 (TDAG51) was induced in both cases. In contrast to their repression by AKT, c-myc, THBS1, and EGR1 were induced by PDGFβ, indicating negative interference between elements upstream and downstream of AKT1 in the PDGFβ signal transduction pathway.
Smooth muscle expression of Cre recombinase and eGFP in transgenic mice
Authors: Xin H.-B. , Deng K.-Y. , Rishniw M. , Ji G. , Kotlikoff M. I.
Published: March 2015

We report the generation of transgenic mice designed to facilitate the study of vascular and nonvascular smooth muscle biology in vivo. The smooth muscle myosin heavy chain (smMHC) promoter was used to direct expression of a bicistronic transgene consisting of Cre recombinase and enhanced green fluorescent protein (eGFP) coding sequences. Animals expressing the transgene display strong fluorescence confined to vascular and nonvascular smooth muscle. Enzymatic dissociation of smooth muscle yields viable, fluorescent cells that can be studied as single cells or sorted by FACS for gene expression studies. smMHC/Cre/eGFP mice were crossed with ROSA26/lacZ reporter mice to determine Cre recombinase activity; Cre recombinase was expressed in all smooth muscles in adult mice, and there was an excellent overlap between expression of the recombinase and eGFP. Initial smooth muscle-specific expression of fluorescence and Cre recombinase was detected on embryonic day 12.5. These mice will be useful to define smooth muscle gene function in vivo in mice, for the study of gene function in single, live cells, and for the determination of gene expression in vascular and nonvascular smooth muscle.
What can cardiovascular gene transfer learn from genomics: and vice versa?
Authors: Kim Tae Ho , Skelding Kimberly A. , Nabel Elizabeth G. , Simari Robert D.
Published: March 2015

The field of gene transfer has developed in an era of expanding biomedical knowledge. The potential for gene transfer to treat cardiovascular disease is great, yet identified and unidentified barriers remain. Gene transfer and its ultimate application, gene therapy, require extensive details of not only the mechanism of disease but the biological implications of the vectors used to deliver the therapeutic genes as well. Many of these details are becoming available via the study of genomics. Genomics, the study of complete genetic sequences, holds the potential for enabling and amplifying the therapeutic hopes for gene transfer. Identification of new therapeutic genes, new regulatory sequences, and establishing the patterns of gene expression from tissues exposed to vectors and transgenes will rapidly advance the application of gene transfer. Finally, there are historical and ongoing lessons learned from the development of gene transfer that may be applicable to the challenging field of genomics and may enable its future success.
Tools for targeted manipulation of the mouse genome
Authors: van der Weyden Louise , Adams David J. , Bradley Allan
Published: March 2015

In the postgenomic era the mouse will be central to the challenge of ascribing a function to the 40,000 or so genes that constitute our genome. In this review, we summarize some of the classic and modern approaches that have fueled the recent dramatic explosion in mouse genetics. Together with the sequencing of the mouse genome, these tools will have a profound effect on our ability to generate new and more accurate mouse models and thus provide a powerful insight into the function of human genes during the processes of both normal development and disease.
Gene expression profiling of human aortic endothelial cells exposed to disturbed flow and steady laminar flow
Authors: Brooks Alan R. , Lelkes Peter I. , Rubanyi Gabor M.
Published: March 2015

Subtraction cloning and cDNA arrays were used to compare steady-state mRNA levels in cultured human aortic endothelial cells (HAEC) exposed for up to 24 h to either high-shear (13 dyn/cm2) steady laminar flow (LF), an established representation of “atheroprotective” flow conditions, or low-shear (<1 dyn/cm2), pulsatile, nonsteady, non-unidirectional flow (disturbed flow, DF) that simulates conditions in the atherosclerosis-prone areas of the arterial circulation. More than 100 genes not previously known to be flow regulated were identified. Analysis of selected genes by quantitative RT-PCR confirmed the results obtained from the microarrays. These data demonstrate that DF is not simply the absence of LF but in fact represents a distinct biomechanical stimulus that has a profound impact upon the gene expression profile of HAEC in culture. In line with previous studies, many of the changes in mRNA levels induced by LF are atheroprotective. In contrast, DF upregulated the mRNA levels of a plethora of proatherosclerotic genes including proinflammatory, proapoptotic, and procoagulant molecules. For some of the genes whose expression was altered by DF and LF, corresponding changes in EC function (proliferation and monocyte adhesion) could be demonstrated. Specifically, the sustained upregulation of VCAM-1 and increased monocyte adhesion to EC exposed to DF was similar to that found in EC in vivo at atherosclerosis-prone regions, confirming the relevance of our model system for in vivo conditions. Distinct differences in the cellular response induced by TNFα and DF suggest that the effects of DF are not mediated entirely by the same signaling pathways that activate NF-κB. These studies demonstrate extensive and pathophysiologically relevant changes in sustained gene expression patterns in aortic EC exposed to DF compared with LF which are predicted to induce a proatherogenic EC phenotype.
Molecular mechanisms of reduced β-adrenergic signaling in the aged heart as revealed by genomic profiling
Authors: Dobson James G. , Fray John , Leonard Jack L. , Pratt Richard E.
Published: March 2015

Myocardial aging leads to a reduction of β-adrenergic receptor-induced metabolic and contractile responsiveness. We hypothesize that a change in the patterns of gene expression is important in these age-related events. To test this, hearts were harvested from young and aged male rats (3–4 and 20–22 mo, respectively). Total mRNA was extracted and prepared for hybridization to Affymetrix U34A GeneChips. Filtering criteria, involving fold change and a statistical significance cutoff were employed, yielding 263 probe pairs exhibiting differential signals. Of the 163 annotated genes, at least 56 (34%) were classified as signaling/cell communication. Of these 56, approximately half were directly involved in G protein-coupled receptor signaling pathways. We next determined which of these changes might be involved in anti-adrenergic activity and identified 19 potentially important gene products. Importantly, we observed a decrease in β1-adrenergic receptor and adenylyl cyclase mRNAs, whereas the mRNA encoding β-arrestin increased. Furthermore, the results demonstrate an increase in mRNAs encoding the adenosine A1 receptor and phospholipase D, which could increase anti-adrenergic effects. Moreover, the mRNAs encoding the muscarinic M3 receptor, nicotinic acetylcholine receptor β3, and nicotinic acetylcholine receptor-related protein were increased as was the mRNA encoding guanylate kinase-associated protein. Interestingly, we also observed eight mRNAs whose abundance changed three- to sixfold with aging that could be considered as being compensatory. Although these results do not prove causality, they demonstrate that cardiac aging is associated with changes in the profiles of gene expression and that many of these changes may contribute to reduced adrenergic signaling.
Optical dysfunction of the crystalline lens in aquaporin-0-deficient mice
Authors: SHIELS ALAN , BASSNETT STEVEN , VARADARAJ KULANDAIAPPA , MATHIAS RICHARD , AL-GHOUL KRISTIN , KUSZAK JER , DONOVIEL DORIT , LILLEBERG STAN , FRIEDRICH GLENN , ZAMBROWICZ BRIAN
Published: March 2015

Aquaporin-0 (AQP0), a water transport channel protein, is the major intrinsic protein (MIP) of lens fiber cell plasma membranes. Mice deficient in the gene for AQP0 ( Aqp0, Mip) were generated from a library of gene trap embryo stem cells. Sequence analysis showed that the gene trap vector had inserted into the first exon of Aqp0, causing a null mutation as verified by RNA blotting and immunochemistry. At 3 wk of age (postnatal day 21), lenses from null mice ( Aqp0−/−) contained polymorphic opacities, whereas lenses from heterozygous mice ( Aqp0+/−) were transparent and did not develop frank opacities until ∼24 wk of age. Osmotic water permeability values for Aqp0+/−and Aqp0−/−lenses were reduced to ∼46% and ∼20% of wild-type values, respectively, and the focusing power of Aqp0+/−lenses was significantly lower than that of wild type. These findings show that heterozygous loss of AQP0 is sufficient to trigger cataractogenesis in mice and suggest that this MIP is required for optimal focusing of the crystalline lens.
Two closely linked interactive blood pressure QTL on rat chromosome 5 defined using congenic Dahl rats
Authors: Garrett Michael R. , Rapp John P.
Published: March 2015

Previously we reported the construction of a congenic strain, S.LEW( 5 ), spanning a large region of rat chromosome 5. The Lewis (LEW) strain was the donor, and the Dahl salt-sensitive (S) strain was the recipient. The congenic strain included a blood pressure quantitative trait locus (QTL). In the present work, a series of nine congenic substrains were constructed from S.LEW( 5 ) which defined two closely linked blood pressure QTL in the region previously thought to contain only one. LEW low-blood-pressure alleles at both QTL were required for a major effect on blood pressure. Neither LEW allele alone had a significant effect on blood pressure. The two QTL were localized to regions 6.3 and 4.6 cM, and these were 1.0 cM apart.
Transcriptional mapping and genomic analysis of the cardiac atria and ventricles
Authors: Zhao Xiao-Song , Gallardo Teresa D. , Lin Ling , Schageman Jeoffrey J. , Shohet Ralph V.
Published: March 2015

The atria and ventricles of the heart have distinct development, structure, and physiology. However, only a few of the genes that underlie the differences between these tissues are known. We used a murine cardiac cDNA microarray to identify genes differentially expressed in the atria and ventricles. The reliability of these findings is supported by highly concordant repetition of hybridization, recognition of previously known atrial and ventricular isoforms of contractile proteins, and confirmation of results by quantitative PCR and in situ hybridization. We examined the most differentially regulated genes for evolutionarily conserved noncoding sequences and found that atrial-expressed genes have more predicted myocyte enhancer factor-2 (MEF2) binding sites than ventricle-predominant genes. We confirmed that messages for MEF2 family members are more abundant in the atria, as are their protein products. Moreover, the activity of a transgenic reporter construct for MEF2 activity is preferentially upregulated in the atria in response to hypertrophic stimuli. This study provides a greater understanding of the molecular differences between atria and ventricles and establishes the framework for an anatomically detailed evaluation of cardiac transcriptional regulation.
Differential myocardial gene expression in the development and rescue of murine heart failure
Authors: Blaxall Burns C. , Spang Rainer , Rockman Howard A. , Koch Walter J.
Published: March 2015

Numerous murine models of heart failure (HF) have been described, many of which develop progressive deterioration of cardiac function. We have recently demonstrated that several of these can be “rescued” or prevented by transgenic cardiac expression of a peptide inhibitor of the β-adrenergic receptor kinase (βARKct). To uncover genomic changes associated with cardiomyopathy and/or its phenotypic rescue by the βARKct, oligonucleotide microarray analysis of left ventricular (LV) gene expression was performed in a total of 53 samples, including 12 each of Normal, HF, and Rescue. Multiple statistical analyses demonstrated significant differences between all groups and further demonstrated that βARKct Rescue returned gene expression toward that of Normal. In our statistical analyses, we found that the HF phenotype is blindly predictable based solely on gene expression profile. To investigate the progression of HF, LV gene expression was determined in young mice with mildly diminished cardiac function and in older mice with severely impaired cardiac function. Interestingly, mild and advanced HF mice shared similar gene expression profiles, and importantly, the mild HF mice were predicted as having a HF phenotype when blindly subjected to our predictive model described above. These data not only validate our predictive model but further demonstrate that, in these mice, the HF gene expression profile appears to already be set in the early stages of HF progression. Thus we have identified methodologies that have the potential to be used for predictive genomic profiling of cardiac phenotype, including cardiovascular disease.
Insights into Dahl salt-sensitive hypertension revealed by temporal patterns of renal medullary gene expression
Authors: Liang Mingyu , Yuan Baozhi , Rute Elizabeth , Greene Andrew S. , Olivier Michael , Cowley Allen W.
Published: March 2015

Dahl salt-sensitive SS and consomic, salt-resistant SS-13BN/Mcw rats possess a highly similar genetic background but exhibit substantial differences in blood pressure salt sensitivity. We used cDNA microarrays to examine sequential changes of mRNA expression of ∼2,000 currently known rat genes in the renal medulla (a tissue critical for long-term blood pressure regulation) in SS and SS-13BN/Mcw rats in response to a high-salt diet (16 h, 3 days, or 2 wk). Differentially expressed genes in each between-group comparison were identified based on a threshold determined experimentally using a reference distribution that was constructed by comparing rats within the same group. A difference analysis of 54 microarrays identified 50 genes that exhibited the most distinct temporal patterns of expression between SS and SS-13BN/Mcw rats over the entire time course. Thirty of these genes could be linked to the regulation of arterial blood pressure or renal injury based on their known involvement in functional pathways such as renal tubular transport, metabolism of vasoactive substances, extracellular matrix formation, and apoptosis. Importantly, the majority of the 30 genes exhibited temporal expression patterns that would be expected to lower arterial pressure and reduce renal injury in SS-13BN/Mcw compared with SS rats. The phenotypic impact of the other 20 genes was less clear. These 50 genes are widely distributed on chromosome 13 and several other chromosomes. This suggested that primary genetic defects, although important, are unlikely to be solely responsible for the full manifestation of this type of hypertension and associated injury phenotypes. In summary, the results of this study identified a number of pathways potentially important for the amelioration of hypertension and renal injury in SS-13BN/Mcw rats, and these results generated a series of testable hypotheses related to the role of the renal medulla in the complex mechanism of salt-sensitive hypertension.
Differential expression of NAT1 translational repressor during development of bovine intramuscular adipocytes
Authors: Childs Kirby D. , Goad David W. , Allan Mark F. , Pomp Daniel , Krehbiel Clinton , Geisert Rodney D. , Morgan J. Brad , Malayer Jerry R.
Published: March 2015

This study was undertaken to test for differential gene expression in intramuscular adipocytes during fat deposition of feedlot steers. Angus × Hereford steers ( n = 50) were fed a high-energy concentrate ration ad libitum for 20 ( n = 5), 86 ( n = 15), 121 ( n = 15), and 146 days ( n = 15) to obtain various degrees of intramuscular adipocyte development. Carcass traits were significantly different ( P < 0.05) between the groups. Intramuscular adipose tissue was excised from the longissimus dorsi and snap frozen in liquid nitrogen. Pooled samples of total RNA representing each group were analyzed by differential-display polymerase chain reaction using 200 primer combinations comprising 20 arbitrary (5′) and 10 anchor (3′) oligonucleotides. Bands ( n = 70) representing putative differences among treatment groups were excised, sequenced, and subjected to BLAST homology search. From these, 40 contained significant homology to known genes. One was of particular interest, the translational repressor NAT1 (novel APOBEC-1 target-1). NAT1 mRNA was quantified in individual animals to confirm differential expression among treatment groups. Results indicate that NAT1 message is more abundant ( P < 0.05) in intramuscular adipocytes of younger/leaner animals.
Abnormal Na channel gating in murine cardiac myocytes deficient in myotonic dystrophy protein kinase
Authors: Lee Hwa C. , Patel Manoj K. , Mistry Dilawaar J. , Wang Qingcai , Reddy Sita , Moorman J. Randall , Mounsey J. Paul
Published: March 2015

DMPK is a serine/threonine kinase implicated in the human disease myotonic muscular dystrophy (DM). Skeletal muscle Na channels exhibit late reopenings in Dmpk-deficient mice and peak current density is reduced, implicating DMPK in regulation of membrane excitability. Since complete heart block and sudden cardiac death occur in the disease, we tested the hypothesis that cardiac Na channels also exhibit abnormal gating in Dmpk-deficient mice. We made whole cell and cell-attached patch clamp recordings of ventricular cardiomyocytes enzymatically isolated from wild-type, Dmpk+/−, and Dmpk−/− mice. Recordings from membrane patches containing one or a few Na channels revealed multiple Na channel reopenings occurring after the macroscopic Na current had subsided in both Dmpk+/− and Dmpk−/− muscle, but only rare reopenings in wild-type muscle (>3-fold difference, P < 0.05). This resulted in a plateau of non-inactivating Na current in Dmpk-deficient muscle. The magnitude of this plateau current was independent on the magnitude of the test potential from −40 to 0 mV and was also independent of gene dose. Macroscopic Na current density was similar in wild-type and Dmpk-deficient muscle, as was steady-state Na channel gating. Decay of macroscopic currents was slowed in Dmpk−/− muscle, but not in Dmpk+/− or wild-type muscle. Entry into, and recovery from, inactivation were similar at multiple test potentials in wild-type and Dmpk-deficient muscle. Resting membrane potential was depolarized, and action potential duration was significantly prolonged in Dmpk-deficient muscle. Thus in cardiac muscle, Dmpk deficiency results in multiple late reopenings of Na channels similar to those seen in Dmpk-deficient skeletal muscle. This is reflected in a plateau of non-inactivating macroscopic Na current and prolongation of cardiac action potentials.
Divergent abnormal muscle relaxation by hypertrophic cardiomyopathy and nemaline myopathy mutant tropomyosins
Authors: Michele Daniel E. , Coutu Pierre , Metzger Joseph M.
Published: March 2015

Mutations in tropomyosin (Tm) have been linked to distinct inherited diseases of cardiac and skeletal muscle, hypertrophic cardiomyopathy (HCM), and nemaline myopathy (NM). How HCM and NM mutations in nearly identical Tm proteins produce the vastly divergent clinical phenotypes of heightened, prolonged cardiac muscle contraction in HCM and skeletal muscle weakness in NM is currently unknown. We report here a direct comparison of the effects of HCM (A63V) and NM (M9R) mutant Tm on membrane-intact myocyte contractile function as assessed by adenoviral gene transfer to fully differentiated cardiac muscle cells. Wild-type, and mutant HCM, and mutant NM proteins were expressed at similar levels in myocytes and incorporated into sarcomeres. Interestingly, HCM mutant Tm produced significantly longer contractions by slowing relaxation, whereas NM mutant Tm produced the opposite effect of accelerated muscle relaxation. We propose slowed relaxation caused by HCM mutant Tm can directly contribute to diastolic dysfunction seen in HCM even without secondary cardiac remodeling. Conversely, hastening of relaxation by NM mutant Tm may shift the force-frequency relationship in skeletal muscle and contribute to muscle weakness seen in NM. Together, these results implicate divergent, abnormal “turning off” of muscle contraction as a cellular basis for the differential pathogenesis of mutant Tm-associated HCM and NM.
Vitamin E deficiency and metabolic deficits in neuronal ceroid lipofuscinosis described by bioinformatics
Authors: Griffin J. L. , Muller D. , Woograsingh R. , Jowatt V. , Hindmarsh A. , Nicholson J. K. , Martin J. E.
Published: March 2015

The mnd mouse, a model of neuronal ceroid lipofusinosis (NCL), has a profound vitamin E deficiency in sera and brain, associated with cerebral deterioration characteristic of NCL. In this study, the vitamin E deficiency is corrected using dietary supplementation. However, the histopathological features associated with NCL remained. With use of a bioinformatics approach based on high-resolution solid and solution state1H-NMR spectroscopy and principal component analysis (PCA), the deficits associated with NCL are defined in terms of a metabolic phenotype. Although vitamin E supplementation reversed some of the metabolic abnormalities, in particular the concentration of phenylalanine in extracts of cerebral tissue, PCA demonstrated that metabolic deficits associated with NCL were greater than any effects produced from vitamin E supplementation. These deficits included increased glutamate and N-acetyl-l-aspartate and decreased creatine and glutamine concentrations in aqueous extracts of the cortex, as well as profound accumulation of lipid in intact cerebral tissue. This is discussed in terms of faulty production of mitochondrial-associated membranes, thought to be central to the deficits in mnd mice.
G protein β3 polymorphism and hemodynamic and body composition phenotypes in the HERITAGE Family Study
Authors: Rankinen Tuomo , Rice Treva , Leon Arthur S. , Skinner James S. , Wilmore Jack H. , Rao D. C. , Bouchard Claude
Published: March 2015

A C825T polymorphism of the G protein β3 ( GNB3) gene has been reported to be associated with hypertension and obesity. We analyzed the associations between the GNB3 C825T polymorphism and hemodynamic and body composition phenotypes in the sedentary state and their responses to endurance training in mainly normotensive white ( n = 473) and black ( n = 255) men and women. Blood pressure (BP) and heart rate (HR) were measured at rest and during submaximal exercise at constant power output (50 W), and stroke volume and cardiac output were obtained during exercise. Body composition was assessed with underwater weighing. Baseline systolic BP (SBP) at 50 W was slightly higher in the white CC homozygotes ( P = 0.036), whereas in blacks the CC genotype was associated with a lower resting HR ( P = 0.012). In blacks, the CC homozygotes showed a greater training-induced reduction in HR at 50 W ( P = 0.013) and a similar trend was observed also in whites ( P = 0.053). Black women carrying the CC genotype showed significantly greater reductions in resting SBP and diastolic BP (DBP) than the TT homozygotes, whereas in black men the changes in resting BP were similar across the genotypes ( P < 0.05 for sex-by- GNB3 interactions). The GNB3 genotype was not associated with baseline body composition in blacks or whites. In blacks, the TT genotype was associated with a greater training-induced decrease in fat mass ( P = 0.012) and percent body fat ( P = 0.006). These data suggest that DNA sequence variation in the GNB3 locus is not a major modifier of endurance training-induced changes in hemodynamic and body composition phenotypes in healthy but previously sedentary subjects. The GNB3 genotype may play a minor role in HR and body fatness regulation in blacks and in responsiveness of resting BP to endurance training in black women.
Effect of hypoxia on gene expression by human hepatocytes (HepG2)
Authors: Sonna Larry A. , Cullivan Michael L. , Sheldon Holly K. , Pratt Richard E. , Lilly Craig M.
Published: March 2015

The full extent to which hypoxia produces gene expression changes in human cells is unknown. We used late-generation oligonucleotide arrays to catalog hypoxia-induced changes in gene expression in HepG2 cells. Five paired sets of cultures were subjected to either control (room air-5% CO2) or hypoxic (1% O2-5% CO2) conditions for 24 h, and RNA was analyzed on an Affymetrix cDNA array containing ∼12,600 sequences. A statistically significant change in expression was shown by 2,908 sequences (1,255 increased and 1,653 decreased). The observed changes were highly concordant with published literature on hypoxic stress but showed relatively little overlap (12–22%) with changes in gene expression that have been reported to occur after heat stress in other systems. Of note, of these 2,908 sequences, only 387 (213 increased and 174 decreased) both exhibited changes in expression of twofold or greater and were highly expressed in at least three of the five experiments. We conclude that the effect of hypoxia on gene expression by HepG2 cells is broad, has a significant component of downregulation, and includes a relatively small number of genes whose response is truly independent of cell and stress type.
Angiotensinogen M235T polymorphism associates with exercise hemodynamics in postmenopausal women
Authors: McCole Steve D. , Brown Michael D. , Moore Geoffrey E. , Ferrell Robert E. , Wilund Kenneth R. , Huberty Andrea , Douglass Larry W. , Hagberg James M.
Published: March 2015

We sought to determine whether the M235T angiotensinogen (AGT) polymorphism, either interacting with habitual physical activity (PA) levels or independently, was associated with cardiovascular (CV) hemodynamics during maximal and submaximal exercise. Sixty-one healthy postmenopausal women (16 sedentary, 21 physically active, and 24 endurance athletes) had heart rate (HR), blood pressure (BP), cardiac output, stroke volume (SV), total peripheral resistance (TPR), and arteriovenous O2 difference (a-vDo2) assessed during 40, 60, 80, and ∼100% of Vo2 max treadmill exercise. Vo2 max did not differ among AGT genotype groups; however, maximal HR was 14 beats/min higher in AGT TT than MM genotype women ( P < 0.05). AGT TT genotype women also had 19 beats/min higher HR during ∼100% Vo2 max exercise than AGT MM genotype women ( P = 0.008). AGT genotype also interacted with habitual PA levels to associate with systolic BP and a-vDo2 during ∼100% Vo2 max exercise (both P < 0.01). AGT TT genotype women had 11 beats/min higher HR during submaximal exercise than MM genotype women ( P < 0.05). AGT genotype interacted with habitual PA levels to associate with systolic BP during submaximal exercise ( P = 0.009). AGT genotype, independently or interacting with habitual PA levels, did not associate significantly with diastolic BP, cardiac output, SV, or TPR during maximal or submaximal exercise. Thus this common genetic variant in the renin-angiotensin system appears to associate, both interactively with habitual PA levels and independently, with HR, systolic BP, and a-vDo2 responses to maximal and submaximal exercise in postmenopausal women.
Isolation and characterization of zebrafish NFE2
Authors: Pratt Stephen J. , Drejer Anna , Foott Helen , Barut Bruce , Brownlie Alison , Postlethwait John , Kato Yasutake , Yamamoto Masayuki , Zon Leonard I.
Published: March 2015

Vertebrate hematopoiesis is regulated by distinct cell-specific transcription factors such as GATA-1 and SCL. Mammalian p45-NFE2 was characterized for its ability to bind the hypersensitive sites of the globin locus control region. NFE2 is a member of a cap’n’collar (CNC) and basic zipper (BZIP) superfamily that regulates gene transcription. It has been implicated in diverse processes such as globin gene expression, oxidative stress, and platelet lineage differentiation. Here, we have isolated the zebrafish ortholog of NFE2. The gene is highly homologous, particularly in the DNA-binding domain. Mapping the zebrafish NFE2 to linkage group 23 establishes a region of chromosomal synteny with human chromosome 12, further suggesting evolutionary conservation. During embryogenesis, the zebrafish gene is expressed specifically in erythroid cells and also in the developing ear. NFE2 expression is lacking in zebrafish mutants that have no hematopoietic cells. An analysis of the sauternes mutant, which carries a mutation in the ALAS-2 gene and thus has defective heme synthesis, demonstrates higher levels of NFE2 expression than normal. This further establishes the block to erythroid differentiation in the sauternes mutant. Our studies demonstrate conservation of the vertebrate genetic program for the erythroid lineage.
Rosiglitazone fails to improve hypertriglyceridemia and glucose tolerance in CD36-deficient BN.SHR4 congenic rat strain
Authors: Seda Ondrej , Kazdova Ludmila , Krenova Drahomira , Kren Vladimir
Published: March 2015

The favorable metabolic effects of thiazolidinediones are supposedly related to the peroxisome proliferator-activated receptor-γ (PPARγ)-driven changes in lipid metabolism, particularly in free fatty acid (FFA) trafficking. The fatty acid translocase CD36 is one of the proposed PPARγ targets to mediate this action. We assessed the effect of rosiglitazone (RSG, Avandia) administration in two inbred rat strains, BN/Cub and BN.SHR4 congenic strain, differing in 10 cM proximal segment of chromosome 4. Rats were fed high-sucrose diet with or without RSG for 1 wk. In BN.SHR4, which carries defective Cd36 allele of SHR origin, RSG failed to improve glucose tolerance (assessed by the oral glucose tolerance test), did not lower triglyceridemia, nor induced increases in epididymal and retroperitoneal adipose tissue weights and adipose tissue glucose utilization, effects observed in BN/Cub. On the other hand, the RSG-treated BN.SHR4 showed lower concentrations of FFA and substantial increase in glycogen synthesis and glucose oxidation in skeletal muscle. Altogether, these results support involvement of CD36 in RSG action, suggesting this pharmacogenetic interaction may be of particular importance in CD36-deficient humans.
Expression profiling reveals metabolic and structural components of extraocular muscles
Authors: Fischer M. Dominik , Gorospe J. Rafael , Felder Edward , Bogdanovich Sasha , Pedrosa-Domellöf F. , Ahima Rexford S. , Rubinstein Neal A. , Hoffman Eric P. , Khurana Tejvir S.
Published: March 2015

The extraocular muscles (EOM) are anatomically and physiologically distinct from other skeletal muscles. EOM are preferentially affected in mitochondrial myopathies, but spared in Duchenne’s muscular dystrophy. The anatomical and pathophysiological properties of EOM have been attributed to their unique molecular makeup: an allotype. We used expression profiling to define molecular features of the EOM allotype. We found 346 differentially expressed genes in rat EOM compared with tibialis anterior, based on a twofold difference cutoff. Genes required for efficient, fatigue-resistant, oxidative metabolism were increased in EOM, whereas genes for glycogen metabolism were decreased. EOM also showed increased expression of genes related to structural components of EOM such as vessels, nerves, mitochondria, and neuromuscular junctions. Additionally, genes related to specialized functional roles of EOM such as the embryonic and EOM-specific myosin heavy chains and genes for muscle growth, development, and/or regeneration were increased. The EOM expression profile was validated using biochemical, structural, and molecular methods. Characterization of the EOM expression profile begins to define gene transcription patterns associated with the unique anatomical, metabolic, and pathophysiological properties of EOM.
Clustering gene expression data using adaptive double self-organizing map
Authors: Ressom Habtom , Wang Dali , Natarajan Padma
Published: March 2015

This paper presents a novel clustering technique known as adaptive double self-organizing map (ADSOM). ADSOM has a flexible topology and performs clustering and cluster visualization simultaneously, thereby requiring no a priori knowledge about the number of clusters. ADSOM is developed based on a recently introduced technique known as double self-organizing map (DSOM). DSOM combines features of the popular self-organizing map (SOM) with two-dimensional position vectors, which serve as a visualization tool to decide how many clusters are needed. Although DSOM addresses the problem of identifying unknown number of clusters, its free parameters are difficult to control to guarantee correct results and convergence. ADSOM updates its free parameters during training, and it allows convergence of its position vectors to a fairly consistent number of clusters provided that its initial number of nodes is greater than the expected number of clusters. The number of clusters can be identified by visually counting the clusters formed by the position vectors after training. A novel index is introduced based on hierarchical clustering of the final locations of position vectors. The index allows automated detection of the number of clusters, thereby reducing human error that could be incurred from counting clusters visually. The reliance of ADSOM in identifying the number of clusters is proven by applying it to publicly available gene expression data from multiple biological systems such as yeast, human, and mouse. ADSOM’s performance in detecting number of clusters is compared with a model-based clustering method.
The graft response to transplantation: a gene expression profile analysis
Authors: Christopher Kenneth , Mueller Thomas F. , DeFina Rachel , Liang Yurong , Zhang Jianhua , Gentleman Robert , Perkins David L.
Published: March 2015

Little is known regarding the graft response to transplantation injury. This study investigates the posttransplantation response of genes that are constitutively expressed in the heart. Constitutive heart and lymph node tissue-restricted gene expression was first analyzed with DNA microarrays. To demonstrate changes following transplantation in genes constitutively expressed in the heart, we performed vascularized murine heart transplants in allogeneic (BALB/c to B6), syngeneic (B6 to B6), and alymphoid (BALB/c-RAG2−/−to B6-RAG1−/−) experimental groups. Temporal induction of genes posttransplant relative to constitutive expression was evaluated with DNA microarrays. Dendrograms and self-organizing maps were generated to determine the dissimilarity between the experimental groups and to identify subsets of differentially expressed genes within the groups, respectively. Expression patterns of selected genes were confirmed by real-time PCR. Biological processes were assigned to genes induced posttransplant using the AnnBuilder package via the Gene Ontology Database. Post-transplant, a shift was noted in genes classified as defense, communication, and metabolism. Our results identify novel components of the graft response to transplantation injury and rejection.
Titin is a candidate gene for stroke volume response to endurance training: the HERITAGE Family Study
Authors: Rankinen Tuomo , Rice Treva , Boudreau Anik , Leon Arthur S. , Skinner James S. , Wilmore Jack H. , Rao D. C. , Bouchard Claude
Published: March 2015

A genome-wide linkage scan for endurance training-induced changes in submaximal exercise stroke volume (ΔSV50) in the HERITAGE Family Study revealed two chromosomal regions (2q31–q32 and 10p11.2) with at least suggestive evidence of linkage among white families. Here we report a further characterization of the quantitative trait locus (QTL) in chromosome 2q31 and provide evidence that titin ( TTN) is likely a candidate gene involved. The original linkage was detected with two markers ( D2S335 and D2S1391), and the QTL covered ∼25 million base pairs (Mb). We added 12 microsatellite markers resulting in an average marker density of one marker per 2.3 Mb. The evidence of linkage increased from P = 0.006 to P = 0.0002 and 0.00002 in the multi- and single-point analyses, respectively. The strongest evidence of linkage was seen with two markers in and near the TTN gene. Transmission/disequilibrium test (TDT) with the same marker set provided evidence for association with one of the TTN markers ( D2S385; P = 0.004). TTN is a major contributor to the elasticity of cardiomyocytes and a key regulator of the Frank-Starling mechanism. Since TTN is the largest gene in the human genome, the challenge is to identify the DNA sequence variants contributing to the interindividual differences in cardiac adaptation to endurance training.
Transcriptomal analysis of failing and nonfailing human hearts
Authors: Steenman M. , Chen Y.-W. , Le Cunff M. , Lamirault G. , Varró A. , Hoffman E. , Léger J. J.
Published: March 2015

Heart failure is a multifactorial disease that may result from different initiating events. To contribute to an improved comprehension of normal cardiac function and the molecular events leading to heart failure, we performed large-scale gene expression analysis of failing and nonfailing human ventricle. Our aim was to define and compare expression profiles of 4 specific pathophysiological cardiac situations: 1) left ventricle (LV) from nonfailing heart; 2) LV from failing hearts affected by dilated cardiomyopathy (DCM); 3) LV from failing hearts affected by ischemic CM (ICM); 4) right ventricle (RV) from failing hearts affected by DCM or ICM. We used oligonucleotide arrays representing ∼12,000 human genes. After stringent numerical analyses using several statistical tests, we identified 1,306 genes with a similar expression profile in all 4 cardiac situations, therefore representative of part of the human cardiac expression profile. A total of 95 genes displayed differential expression between failing and nonfailing heart samples, reflecting a reversal to developmental gene expression, dedifferentiation of failing cardiomyocytes, and involvement of apoptosis. Twenty genes were differentially expressed between failing LV and failing RV, identifying possible candidates for different functioning of both ventricles. Finally, no genes were found to be significantly differentially expressed between failing DCM and failing ICM LV, emphasizing that transcriptomal analysis of explanted hearts results mainly in identification of expression profiles of end-stage heart failure and less in determination of expression profiles of the underlying etiology. Taken together, our data resulted in identification of putative transcriptomal landmarks for normal and disturbed cardiac function.