Jan Monti

Integrated transcriptional profiling and linkage analysis for identification of genes underlying disease

Integration of genome-wide expression profiling with linkage analysis is a new approach to identifying genes underlying complex traits. We applied this approach to the regulation of gene expression in the BXH/HXB panel of rat recombinant inbred strains, one of the largest available rodent recombinant inbred panels and a leading resource for genetic analysis of the highly prevalent metabolic syndrome. In two tissues important to the pathogenesis of the metabolic syndrome, we mapped cis- and trans-regulatory control elements for expression of thousands of genes across the genome. Many of the most highly linked expression quantitative trait loci are regulated in cis, are inherited essentially as monogenic traits and are good candidate genes for previously mapped physiological quantitative trait loci in the rat. By comparative mapping we generated a data set of 73 candidate genes for hypertension that merit testing in human populations. Mining of this publicly available data set is expected to lead to new insights into the genes and regulatory pathways underlying the extensive range of metabolic and cardiovascular disease phenotypes that segregate in these recombinant inbred strains.

Soluble epoxide hydrolase is a susceptibility factor for heart failure in a rat model of human disease

We aimed to identify genetic variants associated with heart failure by using a rat model of the human disease. We performed invasive cardiac hemodynamic measurements in F2 crosses between spontaneously hypertensive heart failure (SHHF) rats and reference strains. We combined linkage analyses with genome-wide expression profiling and identified Ephx2 as a heart failure susceptibility gene in SHHF rats. Specifically, we found that cis variation at Ephx2 segregated with heart failure and with increased transcript expression, protein expression and enzyme activity, leading to a more rapid hydrolysis of cardioprotective epoxyeicosatrienoic acids. To confirm our results, we tested the role of Ephx2 in heart failure using knockout mice. Ephx2 gene ablation protected from pressure overload–induced heart failure and cardiac arrhythmias. We further demonstrated differential regulation of EPHX2 in human heart failure, suggesting a cross-species role for Ephx2 in this complex disease.

Alterations in Blood Pressure and Heart Rate Variability in Transgenic Rats With Low Brain Angiotensinogen

To study whether the brain renin-angiotensin system plays a role in the long-term and short-term control of blood pressure and heart rate variability, we examined in transgenic rats [TGR(ASrAOGEN)] with low brain angiotensinogen levels the 24-hour variation of blood pressure and heart rate. Telemetry recordings were made during basal and hypertensive conditions induced by a low-dose subcutaneous infusion of angiotensin II for 7 days. Short-term blood pressure and heart rate variability were evaluated by spectral analysis, and as a measure of baroreflex sensitivity, the average transfer gain between the pressure and heart rate variations was calculated. During the angiotensin II infusion in control but not TGR(ASrAOGEN) rats, the 24-hour rhythm of blood pressure was inverted (5.8±2 versus −0.4±1.8 mm Hg/group of day-night differences of blood pressure, P <0.05, respectively). In both the control and TGR(ASrAOGEN) rats, the 24-hour heart rate rhythms remained unaltered and paralleled those of locomotor activity. The transfer gain between 0.3 to 0.6 Hz was significantly higher in TGR(ASrAOGEN) than in control rats during control (0.71±0.1 versus 0.35±0.06, P <0.05) but not during angiotensin II infusion (0.6±0.07 versus 0.4±0.1, P >0.05). These results demonstrate that the brain renin-angiotensin system plays an important role in mediating the effects of angiotensin II on the circadian variation of blood pressure. Furthermore, these data indicate that a permanent deficiency in the brain renin-angiotensin system alters the reflex control of heart rate in rats.

Identification of Hypertension-Related Genes Through an Integrated Genomic-Transcriptomic Approach

In search for the genetic basis of hypertension, we applied an integrated genomic-transcriptomic approach to identify genes involved in the pathogenesis of hypertension in the Sabra rat model of salt-susceptibility. In the genomic arm of the project, we previously detected in male rats two salt-susceptibility QTLs on chromosome 1, SS1a (D1Mgh2-D1Mit11; span 43.1 cM) and SS1b (D1Mit11-D1Mit4; span 18 cM). In the transcriptomic arm, we studied differential gene expression in kidneys of SBH/y and SBN/y rats that had been fed regular diet or salt-loaded. We used the Affymetrix Rat Genome RAE230 GeneChip and probed >30 000 transcripts. The research algorithm called for an initial genome-wide screen for differentially expressed transcripts between the study groups. This step was followed by cluster analysis based on 2×2 ANOVA to identify transcripts that were of relevance specifically to salt-sensitivity and hypertension and to salt-resistance. The two arms of the project were integrated by identifying those differentially expressed transcripts that showed an allele-specific hypertensive effect on salt-loading and that mapped within the defined boundaries of the salt-susceptibility QTLs on chromosome 1. The differentially expressed transcripts were confirmed by RT-PCR. Of the 2933 genes annotated to rat chromosome 1, 1102 genes were identified within the boundaries of the two blood pressure QTLs. The microarray identified 2470 transcripts that were differentially expressed between the study groups. Cluster analysis identified genome-wide 192 genes that were relevant to salt-susceptibility and/or hypertension, 19 of which mapped to chromosome 1. Eight of these genes mapped within the boundaries of QTLs SS1a and SS1b. RT-PCR confirmed 7 genes, leaving TcTex1, Myadm, Lisch7, Axl-like, Fah, PRC1-like, and Serpinh1. None of these genes has been implicated in hypertension before. These genes become henceforth targets for our continuing search for the genetic basis of hypertension.

The Role of Wnk4 in Polygenic Hypertension. A Candidate Gene Analysis on Rat Chromosome 10

Abstract—Linkage analyses in experimental crosses of stroke-prone spontaneously hypertensive (SHRSP) and normotensive Wistar-Kyoto (WKY) rats have strongly suggested the presence of quantitative trait loci (QTL) influencing blood pressure and ACE levels on rat chromosome 10, which have been confirmed in multiple independent studies. Analysis of the orthologous region on human chromosome 17 also revealed significant linkage to blood pressure in several populations. Wnk4, a gene previously identified to cause pseudohypoaldosteronism type II, a rare mendelian form of arterial hypertension, is located on human chromosome 17. The hypothesis has been advanced that molecular variants of this gene might contribute to common polygenic forms of hypertension, since Wnk4 is located in a region of conserved synteny that demonstrates an overlap between quantitative trait loci for primary hypertension in humans and rats. In this report, we describe the confirmation of the blood pressure QTL on rat chromosome 10 by congenic approaches, spanning the Wnk4 locus. Comparative analysis of the complete coding sequence of Wnk4 in SHRSP and WKY strains revealed no mutation and demonstrated high conservation between rat and human proteins. Furthermore, comparison of mRNA levels in the kidney showed no differences between SHRSP and WKY. Additionally, we excluded a secondary effect of blood pressure on the transcriptional regulation of Wnk4. Our results fail to support a material contribution of Wnk4 to blood pressure regulation in this model of polygenic hypertension. Thus, Wnk4 is likely not to represent the underlying disease gene for the QTL captured in chromosome 10 congenic animals.

A gene expression analysis in rat kidney following high and low salt intake

Background The effects of salt intake on renal regulation have been investigated for decades. To find new pathways and to demonstrate the utility of oligonucleotide expression arrays, we studied whole kidneys. Methods Eight Sprague–Dawley rats were divided into two groups. One group received a 6% salt (by weight) diet, while the other group received a 0.3%, otherwise identical, salt diet for 7 days. The rats were sacrificed after 7 days and the left kidney was subjected to RNA extraction. Oligonucleotide expression arrays (Affymetrix) were used to determine downregulation and upregulation, comparing high with low salt intake. Four rats from each group were studied separately. Results The experiments were reproducible. Thirty genes were downregulated with the high-salt diet, while 35 genes were upregulated. The renin gene, beta-2 glycoprotein-1, retinol binding protein, annexin VI, and the PTP2C protein tyrosine phosphatase were among the downregulated genes. The angiotensin II receptor type 1B receptor, HMG-CoA reductase, B7 antigen, and the rat calcium channel beta subunit III were among the upregulated genes. Differentially regulated were the p55 subunit (upregulated) and the p50 subunit (downregulated) of the phosphatidyl inositol 3-kinase enzyme complex. We verified our results by selecting a high-salt downregulated gene (renin) and an upregulated gene (B7 antigen) and subjecting these genes to real-time polymerase chain reaction. The results were consistent. Conclusion Oligonucleotide expression arrays can detect novel genes encoding for proteins not generally associated with responses to varied salt intake. Experiments of this nature have substantial limitations and require detailed verification. However, overall, the utility is promising.

Human atrial myosin light chain 1 expression attenuates heart failure.

Most patients with hypertrophic cardiomyopathy and congenital heart diseases express the atrial essential myosin light chains (ALC-1) in their ventricles, replacing the ventricular essential light chains (VLC-1). VLC-1/ALC-1 isoform shift is correlated with increases in cardiac contractile parameters of a transgenic rat model overexpressing hALC-1 in the heart (TGR/hALC-1) compared to normal WKY rats. To investigate, whether the benefical effects of the hALC-1 on cardiac contractility could attenuate contractile failure of the overloaded heart, aortocaval shunt operations of 9-10 weeks old WKY and TGR/hALC-1 were performed. 5 weeks later, both animals groups were sacrificed for analysis of cardiac contraction and transgene expression. Control animals were operated but remained normal body and heart weights. The whole heart contractility parameters were evaluated using the Langendorff heart preparation. Shunt-operated TGR/hALC-1 and WKY rats developed comparable levels of cardiac hypertrophy which was associated with significant reduction of contractile parameters of the Langendorff hearts. However, the decline of cardiac contractility was less pronounced in shunt-operated TGR/hALC-1 compared to shunt-operated WKY. In fact, developed left ventricular pressure as well as maximal velocity of pressure development and relaxation were significantly higher in shunt-operated TGR/hALC-1 as compared to shunt-operated WKY. Expression of hALC-1 was 17 microg/mg whole SDS-protein in control (sham-operated) controls and declined significantly to 14 microg/mg whole SDS-protein in hypertrophied TGR/hALC-1. These results demonstrate that the expression of hALC-1 could have a beneficial effect on the overloaded hypertrophied heart.

Visinin-like protein 1 regulates natriuretic peptide receptor B in the heart

Accumulating evidence indicates that Visinin-like protein-1 (VILIP-1), a member of the family of neuronal calcium sensor proteins (NCS), modulates a variety of processes in extra-neuronal tissues. In this study, we describe VILIP-1 expression in the human heart, rat cardiomyocytes, and H9c2 cells, and demonstrate that VILIP-1 regulates the cell surface localization of natriuretic peptide receptor B (NPR-B). In preparations from failing hearts, we observed VILIP-1 downregulation and reduced NPR-B signalling. In conclusion, VILIP-1 deficiency may be responsible for the reduced efficiency of the natriuretic peptide system in cardiac hypertrophy and heart failure and may therefore serve as pharmacological target.

Assessment of the effect of external counterpulsation on myocardial adaptive arteriogenesis by invasive functional measurements — design of the arteriogenesis network trial 2

BACKGROUND Stimulation of collateral artery growth is a promising therapeutic option for patients with coronary artery disease. External counterpulsation is a non-invasive technique suggested to promote the growth of myocardial collateral arteries via increase of shear stress. The Art.Net.2 Trial tests invasively and functionally for the first time the hypothesis whether a treatment course with external counterpulsation (over 7 weeks) can induce the growth of myocardial collateral arteries. METHODS This study is designed as a prospective, controlled, proof-of-concept study. Inclusion criteria are (1) age 40 to 80 years, (2) stable coronary disease, (3) a residual significant stenosis of at least one epicardial artery and (4) a positive ischemic stress-test for the region of interest. As primary endpoint serves the pressure-derived collateral flow index (CFIp), the invasive gold-standard to assess myocardial collateral pathways. CFIp is determined by simultaneous measurement of mean aortic pressure (Pa, mm Hg), distal coronary occlusive (wedge) pressure (Pw, mm Hg) and central venous pressure (Pv, mm Hg). The index is calculated as CFIp=(Pw-Pv)/(Pa-Pv). The pressure derived fractional flow reserve (FFR) and the index of microcirculatory resistance (IMR) are assessed as secondary invasive endpoints to investigate the effect of ECP on the myocardial vasculature. The non-invasive secondary endpoints include symptoms (CCS and NYHA classification), treadmill-testing and analysis of shear-stress related soluble proteins. CONCLUSIONS The Art.Net.-2 Trial will report within the next months whether direct evidence can be brought that ECP promotes coronary collateral growth in patients with stable angina pectoris.

Medikamentöse Therapie zur Prognoseverbesserung nach akutem Myokardinfarkt

ZusammenfassungDie nach der akuten Therapie des Myokardinfarkts beginnende chronische Therapie ist von großer Bedeutung für die Lebensqualität und Prognose der erkrankten Patienten. Frühere Untersuchungen zeigten, daß in den westlichen, industrialisierten Ländern selbst lange etablierte Therapieprinzipien noch nicht bei allen Patienten eingesetzt werden.In dieser Übersichtsarbeit sind die heute bewiesenen Therapiestrategien zusammen mit den größten relevanten klinischen Studien dargestellt. Die wichtigsten Bausteine der langfristigen Infarkttherapie sind die adrenergen Betarezeptorenblocker, die ACE-Hemmer, die Thrombozytenaggregationshemmer sowie die Lipidsenker. Das Ausmaß der Verbesserung der Überlebenszeit mit diesen Substanzen wird anhand der Diskussion der zugrundeliegenden Studien dargestellt. Weitere medikamentöse, noch nicht bewiesene Ansätze, wie die Angiotensinrezeptorblocker (AT1-Antagonisten) und antioxidative Substanzen, werden diskutiert.AbstractThis review article summarizes the long-term standard therapy for patients with myocardial infarction. The chronic therapy is able to significantly improve quality of life and survival of affected patients. Previous studies showed that in most western countries, the established standard therapy is not given to all patients who would benefit from chronic treatment.The essential parts of today’s myocardial infarction treatment consists of effective beta-blockade, inhibition of the angiotensin-conversion enzyme, inhibition of platelet aggregation and lipid lowering agents. This article reviews the clinical benefits which may be expected from each of these therapeutic approaches. Newer, but not yet proven strategies, like blockade of the angiotensin receptor subtype 1 and treatment with antioxidative agents will be discussed.This review article summarizes the long-term standard therapy for patients with myocardial infarction. The chronic therapy is able to significantly improve quality of life and survival of affected patients. Previous studies showed that in most western countries, the established standard therapy is not given to all patients who would benefit from chronic treatment. The essential parts of todays myocardial infarction treatment consists of effective beta-blockade, inhibition of the angiotensin-conversion enzyme, inhibition of platelet aggregation and lipid lowering agents. This article reviews the clinical benefits which may be expected from each of these therapeutic approaches. Newer, but not yet proven strategies, like blockade of the angiotensin receptor subtype 1 and treatment with antioxidative agents will be discussed.