Christian Paultre

The microRNA Signature in Response to Insulin Reveals Its Implication in the Transcriptional Action of Insulin in Human Skeletal Muscle and the Role of a Sterol Regulatory Element–Binding Protein-1c/Myocyte Enhancer Factor 2C Pathway

OBJECTIVE Factors governing microRNA expressions in response to changes of cellular environment are still largely unknown. Our aim was to determine whether insulin, the major hormone controlling whole-body energy homeostasis, is involved in the regulation of microRNA expressions in human skeletal muscle. RESEARCH DESIGN AND METHODS We carried out comparative microRNA (miRNA) expression profiles in human skeletal muscle biopsies before and after a 3-h euglycemic-hyperinsulinemic clamp, with TaqMan low-density arrays. Then, using DNA microarrays, we determined the response to insulin of the miRNA putative target genes in order to determine their role in the transcriptional action of insulin. We further characterized the mechanism of action of insulin on two representative miRNAs, miR-1 and miR-133a, in human muscle cells. RESULTS Insulin downregulated the expressions of 39 distinct miRNAs in human skeletal muscle. Their potential target mRNAs coded for proteins that were mainly involved in insulin signaling and ubiquitination-mediated proteolysis. Bioinformatic analysis suggested that combinations of different downregulated miRNAs worked in concert to regulate gene expressions in response to insulin. We further demonstrated that sterol regulatory element–binding protein (SREBP)-1c and myocyte enhancer factor 2C were involved in the effect of insulin on miR-1 and miR-133a expression. Interestingly, we found an impaired regulation of miRNAs by insulin in the skeletal muscle of type 2 diabetic patients, likely as consequences of altered SREBP-1c activation. CONCLUSIONS This work demonstrates a new role of insulin in the regulation of miRNAs in human skeletal muscle and suggests a possible implication of these new modulators in insulin resistance.

Mental Stress–Induced Increase in Blood Pressure Is Not Related to Baroreflex Sensitivity in Middle-Aged Healthy Men

The baroreflex that acts to blunt blood pressure (BP) variations through opposite variations in heart rate should limit the BP increase produced by an emotional challenge. However, relations between baroreflex sensitivity and BP reactivity induced by a psychological stress in a large group of adults have never been firmly established. In 280 healthy men, rest (10 minutes) and stress (5 minutes) BP and heart rate were recorded beat to beat by a blood pressure monitor. The mental stress was elicited by a well-standardized computerized version of a word color conflict stress test (Stroop Color Test). Rest and stress baroreflex sensitivity was assessed by the cross-spectral analysis of BP and heart rate and by the sequence method. The stress-induced increase in systolic BP (22.4+/-0.1 mm Hg) was not correlated with resting baroreflex sensitivity but was slightly correlated (r=0.2, P<0.001) with BP variability assessed either by standard deviation or by mid-frequency band spectral power. Our results suggested that a centrally mediated sympathetic stimulation overcame cardiac autonomic regulation and emphasized the role of the sympathetic vasoconstriction in the pressure response at the onset of the stressing stimulation. During the sustained sympathoexcitatory phase, the cardiac baroreflex blunts BP variations but at a lower sensitivity.

ARTERIAL BAROREFLEX CONTROL OF HEART PERIOD IS NOT RELATED TO BLOOD PRESSURE VARIABILITY IN CONSCIOUS HYPERTENSIVE AND NORMOTENSIVE RATS

1. The short‐term (within 30 min periods) and the long‐term (among 30 min periods) variabilities, expressed as variation coefficients, of blood pressure (BP) and heart period (HP) were studied using a computer analysis of BP recordings in freely moving genetically hypertensive (LH), normotensive (LN) and low BP (LL) rats of Lyon strains at ages 5, 9, 21 and 40 weeks. The baroreflex control of HP was estimated with the slope of the linear relationship between systolic BP (SBP) and HP (SBP‐HP slope) computed after phenylephrine and nitroglycerin injections.

Heterogeneous computer network for real-time hemodynamic signal processing

A computer network is described that allows real-time processing, graphical monitoring and off-line analysis of blood pressure, nervous activity and Doppler signals recorded in conscious rats. Real-time processing is performed by an acquisition station using a powerful microprocessor, allowing extraction and storage of several characteristic parameters from each cardiac cycle and real-time graphical monitoring. The experimenter can thereby follow the time evolution of the hemodynamic parameters. Experimental data are sent through the local network to a workstation that ensures off-line processing such as chronograms, histograms and statistical analyses.

Beat-to-beat estimation of windkessel model parameters in conscious rats

A windkessel model was applied on a beat-to-beat basis to evaluate the arterial mechanical characteristics in seven conscious rats. Ascending aortic arterial pressure (AP) and blood flow were recorded during steady-state in basal conditions, during infusions of isoprenaline, sodium nitroprusside, and phenylephrine, and after intravenous atenolol injection. For each cardiac cycle the exponential decay time constant (τ) was estimated from the aortic AP curve, peripheral resistances ( R) were taken as the ratio of mean AP to cardiac output, and systemic arterial compliance ( C) was calculated as τ/ R. In all conditions, mean correlation coefficients of the exponential regression and ∼70% of values in each rat were >0.99, demonstrating the model validity. In all conditions τ and C exhibited a large spontaneous variability over time, and beat-to-beat correlations were high between τ and C (0.83 ± 0.03). C was increased by sodium nitroprusside, decreased by isoprenaline, but not significantly decreased by phenylephrine [5.1 ± 0.2, 3.2 ± 0.3, and 3.9 ± 0.2 μl/mmHg, respectively, vs. 4.2 ± 0.3 μl/mmHg (baseline)]. In conclusion, the windkessel model enables τ and C to be reliably estimated in conscious rats during spontaneous and drug-induced hemodynamic variations.A windkessel model was applied on a beat-to-beat basis to evaluate the arterial mechanical characteristics in seven conscious rats. Ascending aortic arterial pressure (AP) and blood flow were recorded during steady-state in basal conditions, during infusions of isoprenaline, sodium nitroprusside, and phenylephrine, and after intravenous atenolol injection. For each cardiac cycle the exponential decay time constant (tau) was estimated from the aortic AP curve, peripheral resistances (R) were taken as the ratio of mean AP to cardiac output, and systemic arterial compliance (C) was calculated as tau/R. In all conditions, mean correlation coefficients of the exponential regression and approximately 70% of values in each rat were > 0.99, demonstrating the model validity. In all conditions tau and C exhibited a large spontaneous variability over time, and beat-to-beat correlations were high between tau and C (0.83 +/- 0.03). C was increased by sodium nitroprusside, decreased by isoprenaline, but not significantly decreased by phenylephrine [5.1 +/- 0.2, 3.2 +/- 0.3, and 3.9 +/- 0.2 microliters/mmHg, respectively, vs. 4.2 +/- 0.3 microliters/mmHg (baseline)]. In conclusion, the windkessel model enables tau and C to be reliably estimated in conscious rats during spontaneous and drug-induced hemodynamic variations.

Mechanisms of spontaneous baroreflex impairment in Lyon hypertensive rats

This experiment aimed at 1) comparing the spontaneous baroreflex sensitivity (SBRS) in Lyon genetically hypertensive (LH), normotensive (LN), and low blood pressure (LL) rats and 2) assessing some aspects of the mechanisms of its impairment in LH rats. Baroreflex was studied in control animals after an early chronic converting enzyme inhibition with perindopril and after a 4-wk infusion of ANG II in perindopril-treated rats. The SBRS was determined with a previously validated method, using statistical dependence between blood pressure (BP) and heart rate values recorded in freely moving animals. LH rats exhibited high BP, cardiac hypertrophy, and decreased SBRS (LH, 1.3 ± 0.2; LN, 2.5 ± 0.4; LL, 2.2 ± 0.4 beats ⋅ min-1 ⋅ mmHg-1). Perindopril prevented the development of hypertension and cardiac hypertrophy and normalized SBRS. BP rose in LH and LL rats after ANG II infusion, but only LH rats, which developed a cardiac hypertrophy, had an impaired SBRS (LH, 1.1 ± 0.2; LN, 2.5 ± 0.2; LL, 2.8 ± 0.3 beats ⋅ min-1 ⋅ mmHg-1). This impairment was partially reversed by an acute ANG II blockade with losartan. These results demonstrate that high BP does not account for the decreased SBRS in LH rats. SBRS impairment could result either from cardiac hypertrophy or from the direct effect of ANG II on the baroreflex loop.This experiment aimed at 1) comparing the spontaneous baroreflex sensitivity (SBRS) in Lyon genetically hypertensive (LH), normotensive (LN), and low blood pressure (LL) rats and 2) assessing some aspects of the mechanisms of its impairment in LH rats. Baroreflex was studied in control animals after an early chronic converting enzyme inhibition with perindopril and after a 4-wk infusion of ANG II in perindopril-treated rats. The SBRS was determined with a previously validated method, using statistical dependence between blood pressure (BP) and heart rate values recorded in freely moving animals. LH rats exhibited high BP, cardiac hypertrophy, and decreased SBRS (LH, 1.3 +/- 0.2; LN, 2.5 +/- 0.4; LL, 2.2 +/- 0.4 beats . min-1 . mmHg-1). Perindopril prevented the development of hypertension and cardiac hypertrophy and normalized SBRS. BP rose in LH and LL rats after ANG II infusion, but only LH rats, which developed a cardiac hypertrophy, had an impaired SBRS (LH, 1.1 +/- 0.2; LN, 2.5 +/- 0.2; LL, 2.8 +/- 0.3 beats . min-1 . mmHg-1). This impairment was partially reversed by an acute ANG II blockade with losartan. These results demonstrate that high BP does not account for the decreased SBRS in LH rats. SBRS impairment could result either from cardiac hypertrophy or from the direct effect of ANG II on the baroreflex loop.

Blood pressure and heart rate variability changes during cardiac surgery with cardiopulmonary bypass

This study investigated patients undergoing elective cardiac surgery to evaluate the effects of cardiopulmonary bypass (CPB) on the spontaneous variability of mean arterial pressure (MAP) and heart rate (HR). Forty‐one adult patients receiving different cardiovascular system drugs were included in the study. Patients were divided into three groups: no preoperative pharmacological cardiovascular treatment (n = 12), beta‐blocker (BB) (n = 13), and angiotensin‐converting enzyme inhibition (ACEI) (n = 16). MAP was recorded before anaesthesia until the end of surgery. MAP and HR variability was analysed in very low‐ (VLF), low‐ (LF) and high‐frequency bands. The LF spectral component of MAP was observed to decrease in patients under ACEI (−92%) or BB (−87%) following induction of anaesthesia. In addition, during CPB, VLF power decreased in BB group (−67%), and LF power decreased in ACEI group (−77%). Concerning HR, VLF spectral power decreased following anaesthesia in BB group (−74%). In addition, after CPB, VLF power reached lower value in ACEI group than in BB group (P < 0.05). LF spectral power of HR showed a large decrease after CPB in ACEI group (−89%). This study showed that MAP variability did not change during CPB in patients with no preoperative pharmacological cardiovascular treatment, suggesting an unaltered vascular control of MAP. Moreover, the change in LF spectral power of MAP in ACEI and BB groups, suggests that both the renin‐angiotensin and sympathetic systems participate to the genesis of LF variability of MAP.

Local Angiotensin Pathways in Human Carotid Atheroma: Towards a Systems Biology Approach

We will summarize the data we have obtained in human carotid artery concerning the organization of an extended local renin angiotensin aldosterone system and its variations at different stages of atheroma. In a system view, we propose a model where concomitant increase in angiotensin and glucocorticoid signaling is induced and amplified in VSMC while vascular smooth muscle cells transdifferentiate toward a lipid storing phenotype.

Identification of spontaneous cardiac baroreflex episodes at different timescales in rats

To study spontaneous cardiac baroreflex at different timescales, a new method has been developed that identifies such episodes. Mean arterial pressure (MAP) and heart rate (HR) were recorded beat to beat over 1 h in freely moving control ( n = 10) and acutely (1 day before study, n = 7) and chronically (2 wk before study, n = 10) sinoaortic-denervated (SAD) 12- to 14-wk-old male Sprague-Dawley rats. These beat-to-beat time series were successively low-pass filtered seven times and resampled at different time intervals from 0.1 to 6.4 s, allowing different timescales to be scanned. With the use of the Z coefficient, the statistical relationship was estimated for the associations of inverse MAP and HR variations when these inverse MAP and HR variations occurred simultaneously or were time shifted. In control rats and for timescales ≥0.4 s, the highest Zcoefficient(0.38) was obtained when MAP variations preceded inverse HR variations by one sampling interval. The baroreflex origin of this link was demonstrated by its disappearance after acute SAD. In conclusion, this method enabled spontaneous baroreflex episodes to be identified for unusually long timescales without limiting the study to fast, linear, stationary, or oscillating phenomena.To study spontaneous cardiac baroreflex at different timescales, a new method has been developed that identifies such episodes. Mean arterial pressure (MAP) and heart rate (HR) were recorded beat to beat over 1 h in freely moving control (n = 10) and acutely (1 day before study, n = 7) and chronically (2 wk before study, n = 10) sinoaortic-denervated (SAD) 12- to 14-wk-old male Sprague-Dawley rats. These beat-to-beat time series were successively low-pass filtered seven times and resampled at different time intervals from 0.1 to 6.4 s, allowing different timescales to be scanned. With the use of the Z coefficient, the statistical relationship was estimated for the associations of inverse MAP and HR variations when these inverse MAP and HR variations occurred simultaneously or were time shifted. In control rats and for timescales > or = 0.4 s, the highest Z coefficient (0.38) was obtained when MAP variations preceded inverse HR variations by one sampling interval. The baroreflex origin of this link was demonstrated by its disappearance after acute SAD. In conclusion, this method enabled spontaneous baroreflex episodes to be identified for unusually long timescales without limiting the study to fast, linear, stationary, or oscillating phenomena.

1A.03: TRANSCRIPTIONAL NETWORK ASSOCIATED WITH THE CONTRACTILE PHENOTYPE OF SMOOTH MUSCLE CELLS IN HUMAN CAROTID ATHEROSCLEROSIS.

Objective: During atherogenesis, vascular smooth muscle cells (VSMCs) undergo a phenotypic modulation leading to migration and loss of contractility. Here we propose a gene regulatory network specific of the contractile phenotype of the carotid VSMCs from transcriptomic data. Design and method: Human carotid atheroma plaque (ATH, Stary>4) and nearby macroscopically intact tissue (MIT, Stary<3) of 32 patients were analysed by microarrays (Affymetrix HuGene-1.0ST). Histological analysis ensured the large predominance of VSMCs in MIT. Vascular smooth muscle contraction (VSMcontr) involved 119 genes (KEGG database). Transcriptional regulators (TRs) were obtained from Genomatix© and KEGG. Co-expression of TRs and VSMcontr genes was assessed by significant pairwise correlations (p < 10–3) between expression levels across the 32 patients. For each TR, its connecting index (CI) with VSMcontr was obtained from its connectivity, number of its significant (p < 0.001) correlations with the VSMcontr genes, weighted by its expression centile rank. Results: Forty VSMcontr genes were under-expressed (localFDR< 5%) in ATH vs MIT: 11 genes encoding contractile proteins and their kinases/phosphatases, 11 genes encoding receptors and Ca2+/K+ channels, and 18 genes involved in Ca2+ or G-protein signalling. They were taken as the core-VSMcontr gene set. TRs showing the highest CI with core-VSMcontr in MIT that strongly decreased in ATH were taken as representative of the contractile phenotype of VSMCs. Conversely, TRs whose CI with core-VSMContr strongly increased reaching the highest levels in ATH were taken as representative of the synthetic phenotype of VSMCs in ATH. Ninety-one TRs had high positive (CI+) or negative (CI-) connecting index with core-VSMcontr specifically in MIT or ATH: 49 TRs with high CI+ (including NRF1, SRF and THRA) and 16 with high CI- (including HIF1A and STAT1) were MIT-specific, whereas 17 (including ERCC6, PRRX1 and ARID5B) and 9 (including RNF4 and USF1) other TRs had respectively high CI+ and high CI- and were ATH-specific. Conclusions: The regulatory network around core-VSMcontr genes showed clear changes in ATH compared to MIT by reducing the involvement of TRs related to cell contractility and energy metabolism, and increasing that of TRs related to cell dedifferentiation, proliferation and migration or with yet unknown function.