WO2017125406A1

WO2017125406A1 - Methods of predicting left ventricular remodeling in subjects suffering from hypertension

Info

Publication number: WO2017125406A1
Application number: PCT/EP2017/050932
Authority: WO
Inventors: Philippe Rouet; Fatima SMIH ROUET
Original assignee: Institut National de la Sante et de la Recherche Medicale INSERM; Universite Toulouse III Paul Sabatier
Current assignee: Institut National de la Sante et de la Recherche Medicale INSERM; Universite de Toulouse
Priority date: 2016-01-19
Filing date: 2017-01-18
Publication date: 2017-07-27
Anticipated expiration: 2018-07-19

Abstract

The present invention relates to methods of predicting left ventricular remodelling in subjects suffering from hypertension. In particular, the present invention relates to a method of predicting left ventricular remodeling in a subject suffering from hypertension comprising i) determining the mean length of the aliphatic chains of the plasma lipids present in the blood sample obtained from the subject, ii) comparing the mean length determined at step i) with a predetermined reference value and iii) concluding that the subject has or is at risk of having left ventricular remodeling when the mean length determined at step i) is higher than the predetermined reference value or concluding that the subject has not or is not at risk of having left ventricular remodeling when the mean length determined at step i) is lower than the predetermined reference value.

Description

METHODS OF PREDICTING LEFT VENTRICULAR REMODELING IN

SUBJECTS SUFFERING FROM HYPERTENSION

FIELD OF THE INVENTION:

The present invention relates to methods of predicting left ventricular remodelling in subjects suffering from hypertension.

BACKGROUND OF THE INVENTION:

Hypertension is the principal etiology of pathologic left ventricular hypertrophy

(LVH) ¹. Pressure-dependent hemodynamic overload induces left ventricular remodeling as an adaptive response to minimize wall stress. Over time, LVH becomes maladaptive and emerges as a strong and independent risk factor for cardiovascular morbidity (heart failure, coronary artery disease, cerebrovascular accidents, ventricular arrhythmia) and mortality.²'³'⁴'^5,6 Indeed, studies indicate that in 20 ⁷ to 36 ⁸ of chronic hypertensive patients, the myocardium undergoes profound structural remodeling, characterized by enhanced cardiomyocyte growth, increased rate of cardiomyocyte apoptosis, accumulation of fibrosis and microcirculatory changes. This structural remodeling disrupts myocardial excitation- contraction coupling and eventually leads to heart failure.^{9 10} During hypertensive LVH, metabolic changes have been observed in the cardiac muscle, which switches its preference from fatty acids to glucose for ATP generation. Rodent models of spontaneous hypertension ¹¹ and humans with essential hypertension^{12 13} display a decrease in myocardial fatty acids uptake and an increased reliance on glucose and lactate for energy provision. In the acute setting, this shift is considered beneficial to the contractile function because enhanced glucose metabolism allows the heart to cope with the increased workload by producing more ATP per molecule of oxygen consumed.¹⁴ However, chronic hypertension induces the reactivation of the fetal gene program and renders this metabolic remodeling persistent¹⁵. Lastly, in advanced pathological hypertrophy, myocardial glucose metabolism is also decreased due to the development of insulin resistance, leaving the heart unable to generate sufficient ATP to sustain the high workload.¹⁶ Because LVH commonly precedes cardiac failure in patients with hypertension, the early detection of left ventricular (LV) remodeling is warranted to optimize health care for hypertensive patients at risk of developing heart failure. Echocardiography and electrocardiography are presently the prominent tools for LVH diagnosis, though these techniques prove to be time consuming and costly. Several studies have proposed the use of positron electron tomography (PET) scan to detect the early metabolic alterations in myocardial tissue during LVH.^{17 18} However, PET cannot be used for routine detection of LVH because it exposes the patients to ionizing radiation and to radioactive substances and it is an expensive procedure, only available in a minority of medical centers. Circulating biomarkers have also been investigated in hypertensive heart disease (i.e. left ventricular hypertrophy secondary to hypertension): cardiotrophin-1¹⁹, annexin A5²⁰, propeptide of procollagen type I²¹ have been discovered but none of these biomarkers is currently used in clinical practice due to their low sensitivity or lack of specificity.

SUMMARY OF THE INVENTION:

The present invention relates to methods of predicting left ventricular remodelling in subjects suffering from hypertension. In particular the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

In the United States, about 77.9 million (1 out of every 3) adults have high blood pressure. High blood pressure is the primary cause of left ventricular hypertrophy (LVH), which represents a potent prognostic factor for heart failure and cardiovascular mortality. Previous studies have shown an altered metabolic profile in hypertensive patients with LVH. The goal of the inventors was to search for blood metabolomic biomarkers of LVH to provide diagnostic utility in the rapid detection of LVH in large populations of hypertensive individuals. The inventors analyzed plasma ^XH NMR-based metabolomic profiling in 48 hypertensive patients with LVH, 48 hypertensive patients with normal LV size and 24 healthy controls. A multivariate analysis of the spectral data set was performed by using the Partial Least Squares Discriminant Analysis (PLS-DA). Moreover, using transcriptomics, the inventors demonstrated metabolic pathway regulation in leukocytes in a subset of the IBLOMAVED cohort (14 hypertensive patients with LVH compared to 14 hypertensive patients without LVH and 8 controls): genes encoding for glycolytic enzymes were found to be overexpressed in hypertensive patients with LVH. From the ^XH NMR-based metabolomic profiling, discriminant variables were identified as signals coming from methylene (-CH2-) and methyl (-CH3) moieties of aliphatic chains from plasma lipids. The -CH2-/-CH3 ratio which is an indicator of the mean length of the aliphatic lipid chains was significantly higher in the LVH group. The inventors propose the -CH2-/-CH3 ratio from plasma aliphatic lipid chains as a biomarker for left ventricular remodeling in hypertension.

Accordingly, the present invention relates to a method of predicting left ventricular remodeling in a subject suffering from hypertension comprising i) determining the mean length of the aliphatic chains of the plasma lipids present in the blood sample obtained from the subject, ii) comparing the mean length determined at step i) with a predetermined reference value and iii) concluding that the subject has or is at risk of having left ventricular remodeling when the mean length determined at step i) is higher than the predetermined reference value or concluding that the subject has not or is not at risk of having left ventricular remodeling when the mean length determined at step i) is lower than the predetermined reference value.

As used herein, the term "hypertension" or "high blood pressure" refers to a resting blood pressure, as measured with a sphygmomanometer, of greater than 120 mmHg (systolic)/80 mmHg (diastolic). Blood pressure between 121-139/81-89 is considered prehypertension and above this level (140/90 mm Hg or higher) is considered high (hypertension). Both prehypertension and hypertension blood pressure are included in the meaning of "hypertension" as used herein. For example, resting blood pressures of 135 mmHg/87 or of 140 mmHg/90 mmHg are intended to be within the scope of the term "hypertension" even though the 135/87 is within a prehypertensive category. Blood pressures of 145 mm Hg/90 mmHg, 140 mmHg/95 mmHg, and 142 mmHg/93 mmHg are further examples of high blood pressures. It will be appreciated that blood pressure normally varies throughout the day. It can even vary slightly with each heartbeat. Normally, it increases during activity and decreases at rest. It's often higher in cold weather and can rise when under stress. More accurate blood pressure readings can be obtained by daily monitoring blood pressure, where the blood pressure reading is taken at the same time each day to minimize the effect that external factors. Several readings over time may be needed to determine whether blood pressure is high.

As used herein, the term "left ventricular remodeling" has its general meaning in the art and it is used to describe the multiple of changes that occur in cardiac shape, size and composition in response to hypertension. Typically, left ventricular remodeling involves profound structural remodeling, characterized by enhanced cardiomyocyte growth, increased rate of cardiomyocyte apoptosis, accumulation of fibrosis and microcirculatory changes. Left ventricular remodeling leads to left ventricular dysfunction. As used herein, the term "left ventricular dysfunction" refers to a condition in which the left ventricle of the heart is functionally impaired. This impairment of the left ventricular causes the heart to become less efficient in pumping blood throughout the body. Left ventricular dysfunction usually leads to heart failure as well as to other cardiovascular complications. A diagnostic of left ventricular dysfunction can be made by measuring the diminished ejection fraction, mitral valve inflow pattern, pulmonary venous inflow pattern or mitral annular velocity using 2-dimensional echocardiogram coupled with Doppler flow or by performing radionuclide ventriculography, a MRI or a computed tomography using routine techniques known to those skilled the art.

As used herein, the term "Risk" in the context of the present invention, relates to the probability that an event will occur over a specific time period, as in the conversion to left ventricular remodeling, and can mean a subject's "absolute" risk or "relative" risk. Absolute risk can be measured with reference to either actual observation post- measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period. Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no- conversion. "Risk evaluation," or "evaluation of risk" in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another, i.e., from a normal condition to left ventricular remodeling or to one at risk of developing left ventricular remodeling. Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of left ventricular remodeling, either in absolute or relative terms in reference to a previously measured population. The methods of the present invention may be used to make continuous or categorical measurements of the risk of conversion to left ventricular remodeling, thus diagnosing and defining the risk spectrum of a category of subjects defined as being at risk of having left ventricular remodeling. In the categorical scenario, the invention can be used to discriminate between normal and other subject cohorts at higher risk of having left ventricular remodeling. In some embodiments, the present invention may be used so as to discriminate those at risk of having left ventricular remodeling from normal, or those having left ventricular remodeling disease from normal.

As used herein, the term "plasma lipid" designates a lipid present in plasma, including cholesterol and triglycerides carried in the blood. As used herein the term "aliphatic chain" has its general meaning in the art and denotes saturated or non-saturated chains, i.e. chains comprising at least one ethylenic unsaturation and/or optionally at least one acetylenic unsaturation. Thus an aliphatic chain comprises methylene (-CH2-) and methyl (-CH3) moieties and the -CH2-/-CH3 ratio is typically an indicator of the mean length of the aliphatic lipid chains. Methods for determining the mean length of aliphatic lipids (e.g. -CH2-/-CH3 ratio) present in the plasma sample obtained from the subject involves conventional and routine techniques. Typically theses techniques involve are based on RMN analysis as described in the example. In more practical manner, the techniques involve mass spectrometry. As used herein, the term "mass spectrometry" or "MS" refers to an analytical technique to identify compounds by their mass. MS refers to methods of filtering, detecting, and measuring ions based on their m/z. MS technology generally includes (1) ionizing the compounds to form charged species (e.g., ions); and (2) detecting the molecular weight of the ions and calculating their m/z. The compounds may be ionized and detected by any suitable means. A "mass spectrometer" generally includes an ionizer and an ion detector. In general, one or more molecules of interest are ionized, and the ions are subsequently introduced into a mass spectrographic instrument where, due to a combination of magnetic and electric fields, the ions follow a path in space that is dependent upon mass ("m") and charge ("z"). See, e.g., U.S. Pat. No. 6,204,500, entitled "Mass Spectrometry From Surfaces;" U.S. Pat. No. 6,107,623, entitled "Methods and Apparatus for Tandem Mass Spectrometry;" U.S. Pat. No. 6,268,144, entitled "DNA Diagnostics Based On Mass Spectrometry;" U.S. Pat. No. 6,124,137, entitled "Surface-Enhanced Photolabile Attachment And Release For Desorption And Detection Of Analytes;" Wright et al., Prostate Cancer and Prostatic Diseases 2:264-76 (1999); and Merchant and Weinberger, Electrophoresis 21: 1164-67 (2000). In some embodiments, sample is subjected to liquid chromatography before subjected to mass spectrometry. Various methods have been described involving the use of high performance liquid chromatography (HPLC) for sample clean-up prior to mass spectrometry analysis. See, e.g., Taylor et al., Therapeutic Drug Monitoring 22:608-12 (2000) (manual precipitation of blood samples, followed by manual CI 8 solid phase extraction, injection into an HPLC for chromatography on a C18 analytical column, and MS/MS analysis); and Salm et al., Clin. Therapeutics 22 Supl. B:B71-B85 (2000). Commercially available HPLC columns include, but are not limited to, polar, ion exchange (both cation and anion), hydrophobic interaction, phenyl, C-2, C-8, C-18, and polar coating on porous polymer columns. During chromatography, the separation of materials is effected by variables such as choice of eluent (also known as a "mobile phase"), choice of gradient elution and the gradient conditions, temperature, etc.

Typically, the predetermined reference value is a threshold value or a cut-off value. Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. For example, retrospective measurement of the mean length of aliphatic chains of plama lipids in properly banked historical subject samples may be used in establishing the predetermined reference value. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after determining the expression level of the selected peptide in a group of reference, one can use algorithmic analysis for the statistic treatment of the expression levels determined in samples to be tested, and thus obtain a classification standard having significance for sample classification. The full name of ROC curve is receiver operator characteristic curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests. ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1 -specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cut-off values (thresholds or critical values, boundary values between normal and abnormal results of diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate to high. When AUC is higher than 0.9, the accuracy is excellent. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER.SAS, CREATE-ROC.SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.

In some embodiments, when it is concluded that the subject has or is at risk of having left ventricular remodeling, the subject is subsequently administered with a therapeutically effective amount of a drug suitable for the treatment and prevention of left ventricular remodeling. Typically said drug is selected from the group consisting of diuretics, angiotensin converting enzyme (ACE) inhibitors, digoxin (also called digitalis), calcium channel blockers, and beta-blockers. In mild cases, thiazide diuretics, such as hydrochlorothiazide at 25-50 mg/day or chlorothiazide at 250-500 mg/day, are useful. However, supplemental potassium chloride may be needed, since chronic diuresis causes hypokalemis alkalosis. Typical doses of ACE inhibitors include captopril at 25-50 mg/day and quinapril at 10 mg/day. In some embodiments, the subject is administered with an adrenergic beta-2 agonist. An "adrenergic beta-2 agonist" refers to adrenergic beta-2 agonists and analogues and derivatives thereof, including, for example, natural or synthetic functional variants which have adrenergic beta-2 agonist biological activity, as well as fragments of an adrenergic beta-2 agonist having adrenergic beta-2 agonist biological activity. Commonly known adrenergic beta-2 agonists include, but are not limited to, clenbuterol, albuterol, formeoterol, levalbuterol, metaproterenol, pirbuterol, salmeterol, and terbutaline. In some embodiments, the subject is administered with an adrenergic beta-1 antagonist. Adrenergic beta-1 antagonists and adrenergic beta-1 blockers refer to adrenergic beta-1 antagonists and analogues and derivatives thereof, including, for example, natural or synthetic functional variants which have adrenergic beta-1 antagonist biological activity, as well as fragments of an adrenergic beta-1 antagonist having adrenergic beta-1 antagonist biological activity. Commonly known adrenergic beta-1 antagonists include, but are not limited to, acebutolol, atenolol, betaxolol, bisoprolol, esmolol, and metoprolol. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1. Methylene (-CH2-) and methyl (-CH3) moieties of aliphatic chains from plasma lipids ratio (R). Formulae illustrate -CH2-/-CH3 ratios according to the fatty acid chain length. Single line inflexions points represent methylene (-CH2-).

Figure 2. (A) Methylene/methyl ratio in aliphatic lipid chains from plasma lipids of control, hypertensive patients with normal LV size (HTN normal LV size) and hypertensive patients with LV hypertrophy (HTN LVH). The methylene/methyl ratio is significantly higher in the HTN LVH group compared to HTN normal LV size group and control. (B) Plasma cholesterol concentration in control, hypertensive patients with normal LV size (HTN normal LV size) and hypertensive patients with LV hypertrophy (HTN LVH). No difference was found among groups.

Figure 3. Receiver Operating Characteristic (ROC) curve analysis of methylene/methyl ratio in relation to hypertensive group with LVH compared to hypertensive group with normal LV size. The AUC (area under the curve) is 0.703 and the p-value <0.0001.

EXAMPLE:

Material & Methods

Subjects

The study population was enrolled from 2007 to 2009 in the cardiology department of Rangueil Hospital, Toulouse, France, consisted of 2 groups of patients: (1) 48 patients with hypertension and normal LV size and (2) 48 patients with hypertension and LV hypertrophy; 24 healthy controls were also included in the study. For transcriptomic analysis, we selected 14 patients from the first group (patients with hypertension and normal LV size) and 14 patients from the second group (patients with hypertension and LVH), matched for age and cardiovascular risk factors; these were compared to 8 controls from the healthy control group. For ^ΧΗ NMR analysis the entire population was considered. Exclusion criteria included presence of chronic and acute heart failure, others form of cardiomyopathy such as familial obstructive hypertrophic cardiomyopathy, dilated cardiomyopathy, toxic cardiomyopathy, myocardial necrosis, heart rhythm problems, active smoking or smoking cessation less than three years previously, hemopathies, septicemia, hepathopathies and kidney failure requiring dialysis. All hypertensive patients of the study had been receiving pharmacological treatments to lower blood pressure. All subjects underwent a complete cardiac evaluation, including medical history, physical examination, blood pressure measurement and an echocardiography. This research protocol was registered in a clinical database (ClinicalTrials.gov NCTO 1024049) and conforms to the ethical guidelines of the 1975 Declaration of Helsinki. The protocol was approved by the institution's human research (COSSEC) and regional ethics committee (Comite de Protection des Personnes (CPP) # DC2008-452). Written informed consent was obtained from all participants.

Blood pressure measurement

All subjects underwent a 24-h ambulatory blood pressure measurement recorded with validated ambulatory recorders (TM 2420 and TM 2421, A & D Engineering, CA, USA).²⁸ A trained nurse fitted a cuff on the left arm of the patients. Monitors were programmed to record blood pressure every 30 min. Patients completed a diary card and pressed the event marker to identify sleep-wake cycle. Patients with 24-h average blood pressure > 140/85 mmHg were defined as hypertensive.

Echocardiography

All subjects underwent complete M-mode, 2-dimensional, and Doppler echocardiography to determine left ventricular ejection fraction (LVEF), left ventricular mass (LVM), and valve function. The LVEF was calculated according to the modified Simpson's rule. The LV mass was indexed to the body surface area to derive the LV mass index (LVMI, g/m²). Left ventricular hypertrophy (LVH) was defined according to the previously established criterion (LVMI >125 g/m² for men and LVMI >110 g/m² for women)²⁹.

Blood sampling

Venous blood samples were collected after overnight fasting in Becton Dickinson Vacutainer CPT tubes with sodium heparin. The blood plasma was immediately separated by centrifugation (3000 RCF/4°C/ 10 min) and aliquots were stored at -80°C. For white blood cell separation, blood samples were collected in 8 ml BD CPT vacutainer tubes and processed immediately according to the manufacturer's protocol. Microarray analysis

Total RNA was purified from collected white blood cells using the RNeasy kit (Qiagen) in a QIAcube automated sample prep system (Qiagen). Total RNA integrity was checked by capillary electrophoresis (Experion, Bio-Rad). Samples with RNA Quality Indicator >8.5/10 were selected for analysis. Total RNAs were quantified with RiboGreen on a VictorTM X5 2030 multilabel reader (Perkin Elmer). A 300 ng aliquot was used for fluorescent labeling (QuickAmp Labeling, Agilent). A pooled reference sample was used as a common reference for all hybridizations. Labeled RNAs were hybridized to Agilent human pangenomic glass microarray. After standard hybridization, the glass arrays were washed in a Ventana robotized apparatus and scanned using a GenePix 4000 scanner (Axon). Scanned images were processed by X-dot reader software with operator validation. Microarray data were deposited in the GEO database (#GSE74144: link for reviewers http://www.ncbi.nlm.nih. gov/geo/query/acc.cgi?token=clyzccygvtenvcl&acc=GSE74144) and followed MIAME requirements. NMR analysis

NMR analysis was performed as previously reported.³⁰

Cholesterol assay

Total plasma cholesterol was measured by the CHOD-PAP method with kit Al 1A01634 (HORIBA ABX diagnostic, Montpellier, France).

Statistical analysis

Data analysis for microarray analysis was performed with «R» software (version 2.14.1) with the bioconductor (http://www.bioconductor.org/) packages. Following quality control and removal of the outliers, data were quantile normalized for each condition. Significant genes were identified using the limma package.³¹ An FDR (False Discovery Rate) corrected p-value <0.05 was used as a criterion for significance. For matabolomics, ROC curve analysis was performed using MedCalc 15.8 software (www.medcalc.be). Continuous variables are presented as mean + SD and categorical as numbers and percentages. Continuous variables were compared with the use of one way-Anova or Kruskal-Wallis rank sum test when normality or equal variance test failed. Categorical variables were compared with the use of the Pearson chi-square (sigma stat). Results

Study population

The patients' characteristics including age, sex, body mass index (BMI), presence of diabetes, dyslipidemia, obesity and echocardiographic parameters are shown in Table 1. The hypertensive population had a systolic blood pressure of 149 + 20 mmHg versus 125 + 13 mmHg for the control group. Patients with LV Hypertrophy displayed a LV mass index of 115 + 33 g/m² and an LV thickness/radius ratio (h/r) of 52 + 8, both significantly greater (p<0.001) when compared to hypertensive patients with no LV remodeling (81 + 18 g/m² and 37 + 4, respectively) and to healthy controls (74 + 18 g/m² and 36+ 5, respectively). Left ventricular ejection fraction was not different among the groups with a value of 69 + 7 % for healthy controls and hypertensive patients with normal LV size and 69 % + 9 for the LVH group. There was no difference between the two hypertensive groups for the prevalence of diabetes, obesity or dyslipidemia. Transcriptomic profiling in white blood cells

The transcriptomic study compared gene expression in leukocytes from 14 hypertensive patients with LVH, 14 hypertensive patients with normal LV size, and 8 controls. The total number of probes tested on the microarray was 25,000. We found 96 genes differentially expressed when comparing hypertensive patients with LVH to hypertensive individuals with normal LV size. Among those, we had a focus on genes involved in metabolism. Four genes encoding glycolytic enzymes were found overexpressed in patients with LV remodeling: hexokinase 1 (HK1), glucose-6-phosphate-isomerase (GPI), glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK1). We also found three genes encoding enzymes involved in energy production that were overexpressed in the white blood cells of hypertensive patients with LVH (data available in GEO database): isocitrate dehydrogenase 2 NADPH dependent (IDH2), isocitrate dehydrogenase NAD⁺ gamma (IDH3G) and ATP synthase H⁺ transporting mitochondrial Fl complex, beta polypeptide (ATP5B). Lastly, two enzymes belonging to the beta-oxidation pathway were also overexpressed in the hypertensive patients with LVH: peroxisomal enoyl- Co A hydratase 1 (ECH1) and the hydroxyacyl-CoA dehydrogenase/3ketoacyl-CoA thiolase/enoylCoA Hydratase trifunctional protein (HADHA) alpha subunit. No changes were found for peroxisome proliferator-activated receptor a (PPARa), master transcriptional regulator of the fatty acid oxidation pathway, or for carnitine palmitoyltransferase IB (CPT- IB), master regulator of long-chain fatty acid entry inside the mitochondria. There was no significant difference in gene expression profiles between the hypertensive group with normal LV size and the control group. Glycolytic enzymes and ECH1 were also found overexpressed in hypertensive patients with LV remodeling when compared to control individuals.

¹H NMR metabolomic profiling of patients' plasmas

Data extracted from the ^XH NMR spectra of the patients' plasma were analyzed using partial least squares discriminant analysis (PLS-DA). PLS-DA score plot of spectral data revealed sample clustering according to the clinical status. In the loading plot, discriminant variables from hypertensive patients with LVH are located in the lower left corner, while the ones from hypertensive patients with normal LV size and healthy subjects are in the upper right corner. Signals in the organic fraction came from a peak at 1.3ppm (part per million) for the hypertensive patients with LVH, while the signals for hypertensive patients with no LVH and healthy controls came from a peak at 0.9ppm. These signals were identified based on their chemical shift and correspond to protons located in the methylene (-CH2-) group for the peak at 1.3ppm and in the methyl (-CH3) group for the peak at 0.9ppm. Both chemical groups are derived from the aliphatic chains of plasma lipids (Figure 1). The methylene/methyl ratio was found to be significantly higher in hypertensive patients with LVH when compared to hypertensive patients with normal LV size or to control individuals (Figure 2A). The methylene/methyl ratio was similar between hypertensive patients with normal LV size and control individuals.

Cholesterol level

There was no difference between the three groups for cholesterol plasma levels (Figure 2B).

Methylene/methyl ratio as predictor of left ventricular hypertrophy

The predictive value of the methylene/methyl ratio for left ventricular hypertrophy was evaluated using ROC curve analysis (Figure 3). The accuracy was estimated at AUC: 0.703, with a p-value <0.0001. Discussion

Prolonged hypertension may result in LV structural remodeling, alteration of cardiac function and chronic heart failure.² Early detection and treatment of LV hypertrophy in hypertensive patients is important in order to prevent the progression towards advanced cardiac disease. While the microarray technique has already been used to report large-scale gene expression changes in leukocytes of patients with essential hypertension²⁴, such study had not been performed yet in hypertensive patients with LVH. We identified changes in the expression of genes controlling metabolic pathways in leukocytes of hypertensive patients with LV remodeling compared to hypertensive patients with normal LV size and control individuals. We sought to determine whether these changes reflect alterations previously reported by PET scan studies of the human heart,¹⁷ or whether they correspond to previously unreported alterations. Interestingly, among the differentially expressed genes, four genes encoding enzymes catalyzing glycolytic steps were found to be specifically overexpressed in hypertensive patients with LV remodeling: hexokinase 1 (HK1), glucose-6-phosphate isomerase (GPI), glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK1). The data is in accordance with previous evidence reporting accelerated glycolytic rates in the hypertrophic heart.^12,32,33 These results also confirm the well established return to a fetal metabolic profile of the hypertrophic heart, with an increased preference for glucose as substrate for energy provision.^34,35,36 Accordingly, we found that among the four known different isoforms of hexokinases, HK1, which is the predominant isoform in the embryonic, fetal, and neonatal hearts^37,38, was specifically overexpressed. Glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and PGK1 are major regulatory enzymes in the glycolytic pathway catalyzing the generation of NADH from NAD⁺ in the sixth step of glycolysis and the first ATP-generating step of the glycolytic pathway, respectively. The expression of GAPDH has been shown to be higher in the fetal heart and increased with cardiac hypertrophy.³⁹ Increased activity of several glycolytic enzymes was also reported in the hypertrophied heart^11,40, even though another study suggested it is not a prerequisite for increased glycolytic flux.⁴¹ Such discrepancies among studies are likely due to differences in disease models and/or in the stage of hypertrophy studied. In addition, our study demonstrates increased expression of genes encoding other enzymes involved in energy production: IDH2 and IDH3G, both are enzymes that participate in the production of NADH in the Krebs cycle and ATP5B is a subunit of the mitochondrial ATP synthase. These changes most likely reflect an increased need for energy provision in the hypertrophied heart of hypertensive patients, because the heart requires more energy to sustain its own enlargement. Utilization of myocardial fatty acids is decreased in the remodeled myocardium of spontaneously hypertensive rats¹¹ and patients with essential hypertension.¹⁷ This phenomenon has been linked to alterations in the activity of PPARa and of its main target genes⁴². Our microarray analysis of white blood cells suggests that the expression of PPARa and of its target genes was unaltered in the heart of hypertensive patients with left ventricular remodeling. Nevertheless, it remains possible that PPARa protein levels were decreased in hypertensive patients with cardiac hypertrophy and this in spite of unchanged mRNA levels as previously reported by others.⁴² We found increased expression of the alpha subunit of the mitochondrial trifunctional protein HADHA, a multienzyme complex involved in mitochondrial long-chain fatty acid beta- oxidation and of peroxisomal ECH1, an enzyme from the peroxisomal beta-oxidation. Increased expression of mitochondrial long-chain enoyl- CoA hydratase in mouse heart has been reported to follow the induction of cardiac hypertrophy.⁴³ Our results are consistent with the concept that the down-regulation of the enzymes controlling fatty acid oxidation occurs in an advanced stage of heart failure, while an increase of glycolysis occurs in the early stage of remodeling.^32,44 Interestingly, the gene expression pattern observed in white blood cells from hypertensive patients with LVH included genes previously involved in cardiac remodeling such as TEVIP metallopeptidase 1 and 2 ⁴⁵ and methionine sulfoxide reductase Bl (MSRB1).⁴⁶ This observation further supports the notion that the transcriptome of white blood cells remodels in parallel to the cardiac transcriptome. In accordance with another study, we found no differences in the transcriptomes of white blood cells between the hypertensive and control groups²⁴. On the other hand, hypertensive patients with LVH displayed a specific pattern of gene expression different from both control and hypertensive patients. Among the genes differentially expressed in those groups, we found again that genes encoding glycolytic enzymes were upregulated, while genes encoding enzymes from the β-oxidation pathway were unchanged. In addition to confirming the clinical status of the patients included in this study, the results once again demonstrate that the transcriptome of white blood cells from hypertensive patients could be used as a fingerprint of left ventricular remodeling.

We then looked for plasma biomarkers of left ventricular remodeling in hypertensive patients using metabolomics. Discriminant variables were identified as signals coming from the methylene and methyl moieties of aliphatic chains from plasma lipids. The methylene/methyl ratio, which is an indicator of the mean aliphatic lipid chains length, was increased in the plasma of hypertensive patients with left ventricular remodeling. Long-chain fatty acids are the preferred substrate for energy provision in the normal heart. The higher content of long-chain fatty acids in the plasma of hypertensive patients with LVH could reflect an impairment of their utilization at the heart level. Indeed, patients with very long- chain acyl-CoA dehydrogenase (VLCAD) deficiency, the enzyme responsible for the catabolism of fatty acids with an acyl chain length between 14 and 18 carbons, exhibit cardiac hypertrophy⁴⁷. Moreover, blood profiles from animals and patients with VLCAD deficiency are characterized by large accumulations of long chain acyl carnitines^48,49. Several investigations in spontaneously hypertensive rats^11,32,50 and in humans with essential hypertension¹⁷ proposed a direct link between decreased myocardial fatty acid transport and the pro-hypertrophic response in the heart. A recent study found that the heart of hypertensive humans expresses a truncated form of PPARa. Increased expression of the truncated PPARa is associated with decreased expression of the PPARa target genes involved in fatty acid transport and oxidation.¹³ Moreover, decreased PPARa expression and/or activity in the heart could be linked to the development of local inflammation and fibrosis.⁵¹ The inhibition of myocardial fatty acid metabolism has been proposed as an independent predictor of the cardiac hypertrophic response in hypertensive patients.¹⁷ In leukocytes, we did not find a decrease in the expression of genes encoding for fatty acid oxidation enzymes, suggesting that their downregulation occurs in a later phase of hypertensive heart disease (i.e. when left ventricular ejection fraction is decreased), and that other molecular mechanisms are likely to be involved in the progression from hypertension alone to hypertension with left ventricular remodeling. The absence of other physiological differences between the two hypertensive groups (no differences in blood cholesterol levels) in our study reinforces the possibility that the methylene/methyl ratio may be used as a diagnostic tool in large screenings for hypertensive left ventricular remodeling. Perspectives of the study

LVH is present in about 35 % of hypertensive individuals. There is a need for a quick, non-invasive and inexpensive method for the early diagnosis of LVH in hypertensive patients (i.e. before alteration of the cardiac function) in order to prevent the progression towards advanced cardiac failure. Gene expression profiling allows a better understanding of pathophysiological processes and may thereby contribute to a better prediction of clinical outcome. Gene expression fingerprinting is still a challenge in the field of cardiology, even though it is becoming a standard procedure in cancerology.⁵² This is due to the difficulty in obtaining heart tissue samples, and because RNA extractions and microarray analyses are difficult to implement for high-throughput screening of patients in clinic. Our study proposes a new circulating biomarker as an alternative to molecular gene expression analysis for the detection of LV remodeling in hypertensive patients.

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Claims

CLAIMS:

1. A method of predicting left ventricular remodeling in a subject suffering from hypertension comprising i) determining the mean length of the aliphatic chains of the plasma lipids present in the blood sample obtained from the subject, ii) comparing the mean length determined at step i) with a predetermined reference value and iii) concluding that the subject has or is at risk of having left ventricular remodeling when the mean length determined at step i) is higher than the predetermined reference value or concluding that the subject has not or is not at risk of having left ventricular remodeling when the mean length determined at step i) is lower than the predetermined reference value.

2. The method of claim 1 wherein the mean length of the aliphatic chains of the plasma lipids is determined by mass spectrometry.

3. The method of claim 1 wherein when it is concluded that the subject has or is at risk of having left ventricular remodeling, the subject is subsequently administered with a therapeutically effective amount of a drug suitable for the treatment and prevention of left ventricular remodeling.

4. The method of claim 3 wherein the drug is selected from the group consisting of diuretics, angiotensin converting enzyme (ACE) inhibitors, digoxin (also called digitalis), calcium channel blockers, adrenergic beta-1 antagonist and beta-blockers.