Global longitudinal strain and plasma biomarkers for prognosis in heart failure complicated by diabetes: a prospective observational study.

BACKGROUND: Heart failure (HF) and diabetes are associated with increased incidence and worse prognosis of each other. The prognostic value of global longitudinal strain (GLS) measured by cardiovascular magnetic resonance (CMR) has not been established in HF patients with diabetes. METHODS: In this prospective, observational study, consecutive patients (n = 315) with HF underwent CMR at 3T, including GLS, late gadolinium enhancement (LGE), native T1, and extracellular volume fraction (ECV) mapping. Plasma biomarker concentrations were measured including: N-terminal pro B-type natriuretic peptide(NT-proBNP), high-sensitivity troponin T(hs-TnT), growth differentiation factor 15(GDF-15), soluble ST2(sST2), and galectin 3(Gal-3). The primary outcome was a composite of all-cause mortality or HF hospitalisation. RESULTS: Compared to those without diabetes (n = 156), the diabetes group (n = 159) had a higher LGE prevalence (76 vs. 60%, p < 0.05), higher T1 (1285±42 vs. 1269±42ms, p < 0.001), and higher ECV (30.5±3.5 vs. 28.8±4.1%, p < 0.001). The diabetes group had higher NT-pro-BNP, hs-TnT, GDF-15, sST2, and Gal-3. Diabetes conferred worse prognosis (hazard ratio (HR) 2.33 [95% confidence interval (CI) 1.43-3.79], p < 0.001). In multivariable Cox regression analysis including clinical markers and plasma biomarkers, sST2 alone remained independently associated with the primary outcome (HR per 1 ng/mL 1.04 [95% CI 1.02-1.07], p = 0.001). In multivariable Cox regression models in the diabetes group, both GLS and sST2 remained prognostic (GLS: HR 1.12 [95% CI 1.03-1.21], p = 0.01; sST2: HR per 1 ng/mL 1.03 [95% CI 1.00-1.06], p = 0.02). CONCLUSIONS: Compared to HF patients without diabetes, those with diabetes have worse plasma and CMR markers of fibrosis and a more adverse prognosis. GLS by CMR is a powerful and independent prognostic marker in HF patients with diabetes.

A porcine model of heart failure with preserved ejection fraction: magnetic resonance imaging and metabolic energetics.

AIMS: A significant proportion of heart failure (HF) patients have HF preserved ejection fraction (HFpEF). The lack of effective treatments for HFpEF remains a critical unmet need. A key obstacle to therapeutic innovation in HFpEF is the paucity of pre-clinical models. Although several large animal models have been reported, few demonstrate progression to decompensated HF. We have established a model of HFpEF by enhancing a porcine model of progressive left ventricular (LV) pressure overload and characterized HF in this model including advanced cardiometabolic imaging using cardiac magnetic resonance imaging and hyperpolarized carbon-13 magnetic resonance spectroscopy. METHODS AND RESULTS: Pigs underwent progressive LV pressure overload by means of an inflatable aortic cuff. Pigs developed LV hypertrophy (50% increase in wall thickness, P < 0.001, and two-fold increase in mass compared to sham control, P < 0.001) with no evidence of LV dilatation but a significant increase in left atrial volume (P = 0.013). Cardiac magnetic resonance imaging demonstrated T1 modified Look-Locker inversion recovery values increased in 16/17 segments compared to sham pigs (P < 0.05-P < 0.001) indicating global ventricular fibrosis. Mean LV end-diastolic (P = 0.047) and pulmonary capillary wedge pressures (P = 0.008) were elevated compared with sham control. One-third of the pigs demonstrated clinical signs of frank decompensated HF, and mean plasma BNP concentrations were raised compared with sham control (P = 0.008). Cardiometabolic imaging with hyperpolarized carbon-13 magnetic resonance spectroscopy agreed with known metabolic changes in the failing heart with a switch from fatty acid towards glucose substrate utilization. CONCLUSIONS: Progressive aortic constriction in growing pigs induces significant LV hypertrophy with cardiac fibrosis associated with left atrial dilation, raised filling pressures, and an ability to transition to overt HF with raised BNP without reduction in LVEF. This model replicates many aspects of clinical HFpEF with a predominant background of hypertension and can be used to advance understanding of underlying pathology and for necessary pre-clinical testing of novel candidate therapies.

Multimodal assessments of Zika virus immune pathophysiological responses in marmosets.

Animal models that recapitulate the human pathophysiology have been developed as useful research tools. Although laboratory mice are widely used, they are phylogenetically "distant" to humans. New world monkeys, such as the common marmoset (Callithrix jacchus) have steadily gained prominence. In this report, marmosets are explored as an alternate in vivo model to investigate infection and immunity of Zika virus (ZIKV). Multimodal platforms, including ultrasound and magnetic resonance imaging (MRI), flow cytometry, and multiplex microbead immunoassays were established to comprehensively decipher immune responses and pathophysiological outcomes. While ZIKV-infected marmosets had detectable ZIKV RNA load in various body fluids, animals did not develop any observable lesions in their testes and brains as shown by ultrasound and MRI. Immune-phenotyping detected differences in the numbers of B cells, CD8+ T cells and HLADR+ NK cells during the first two weeks of infection. Neutralizing ZIKV-specific antibodies were elicited to high levels and targeted epitopes in the E protein. This study presents a one-stop-shop platform to study infection and pathophysiology in marmosets. While marmoset-specific research tools are being refined, the research values of these animals present them as a good model for immune-based therapies.

Expression of Cell-Surface Marker ABCB5 Causes Characteristic Modifications of Glucose, Amino Acid and Phospholipid Metabolism in the G3361 Melanoma-Initiating Cell Line.

We present a pilot study aimed at determining the effects of expression of ATP-binding cassette member B5 (ABCB5), a previously described marker for melanoma-initiating cells, on cellular metabolism. Metabolic profiles for two groups of human G3361 melanoma cells were compared, i.e. wildtype melanoma cells with intact ABCB5 expression (ABCB5-WT) and corresponding melanoma cell variants with inhibited ABCB5 expression, through shRNA-mediated gene knockdown (ABCB5-KD). A comprehensive metabolomic analysis was performed by using proton and phosphorus NMR spectroscopy of cell extracts to examine water-soluble metabolites and lipids. Parametric and non-parametric statistical analysis of absolute and relative metabolite levels yielded significant differences for compounds involved in glucose, amino acid and phospholipid (PL) metabolism. By contrast, energy metabolism was virtually unaffected by ABCB5 expression. The sum of water-soluble metabolites per total protein was 17% higher in ABCB5-WT vs. ABCB5-KD G3361 variants, but no difference was found for the sum of PLs. Enhanced abundance was particularly pronounced for lactate (+ 23%) and alanine (+ 26%), suggesting an increase in glycolysis and potentially glutaminolysis. Increases in PL degradation products, glycerophosphocholine and glycerophosphoethanolamine (+ 85 and 123%, respectively), and redistributions within the PL pool suggested enhanced membrane PL turnover as a consequence of ABCB5 expression. The possibility of glycolysis modulation by an ABCB5-dependent IL1ß-mediated mechanism was supported by functional studies employing monoclonal antibody (mAb)-dependent ABCB5 protein inhibition in wildtype G3361 melanoma cells. Our metabolomic results suggest that the underlying biochemical pathways may offer targets for melanoma therapy, potentially in combination with other treatment forms.

Creatine, Glutamine plus Glutamate, and Macromolecules Are Decreased in the Central White Matter of Premature Neonates around Term.

Preterm birth represents a high risk of neurodevelopmental disabilities when associated with white-matter damage. Recent studies have reported cognitive deficits in children born preterm without brain injury on MRI at term-equivalent age. Understanding the microstructural and metabolic underpinnings of these deficits is essential for their early detection. Here, we used diffusion-weighted imaging and single-voxel 1H magnetic resonance spectroscopy (MRS) to compare brain maturation at term-equivalent age in premature neonates with no evidence of white matter injury on conventional MRI except diffuse excessive high-signal intensity, and normal term neonates. Thirty-two infants, 16 term neonates (mean post-conceptional age at scan: 39.8±1 weeks) and 16 premature neonates (mean gestational age at birth: 29.1±2 weeks, mean post-conceptional age at scan: 39.2±1 weeks) were investigated. The MRI/MRS protocol performed at 1.5T involved diffusion-weighted MRI and localized 1H-MRS with the Point RESolved Spectroscopy (PRESS) sequence. Preterm neonates showed significantly higher ADC values in the temporal white matter (P<0.05), the occipital white matter (P<0.005) and the thalamus (P<0.05). The proton spectrum of the centrum semiovale was characterized by significantly lower taurine/H2O and macromolecules/H2O ratios (P<0.05) at a TE of 30 ms, and reduced (creatine+phosphocreatine)/H2O and (glutamine+glutamate)/H2O ratios (P<0.05) at a TE of 135 ms in the preterm neonates than in full-term neonates. Our findings indicate that premature neonates with normal conventional MRI present a delay in brain maturation affecting the white matter and the thalamus. Their brain metabolic profile is characterized by lower levels of creatine, glutamine plus glutamate, and macromolecules in the centrum semiovale, a finding suggesting altered energy metabolism and protein synthesis.

Mitochondrial Coupling and Contractile Efficiency in Humans with High and Low V˙O2peaks.

INTRODUCTION: Endurance training elicits tremendous adaptations of the mitochondrial energetic capacity. Yet, the effects of training or physical fitness on mitochondrial efficiency during exercise are still unclear. Accordingly, the purpose of the present study was to examine in vivo the differences in mitochondrial efficiency and ATP cost of contraction during exercise in two groups of adults differing in their aerobic capacity. METHOD: We simultaneously assessed the ATP synthesis and O2 fluxes with P-magnetic resonance spectroscopy and pulmonary gas exchange measurements in seven endurance-trained (ET, V˙O2max: 67 ± 8 mL·min⁻¹·kg⁻¹) and seven recreationally active (RA, V˙O2max: 43 ± 7 mL·min⁻¹·kg⁻¹) subjects during 6 min of dynamic moderate-intensity knee extension. RESULTS: The ATP cost of dynamic contraction was not significantly different between ET and RA (P > 0.05). Similarly, end-exercise O2 consumption was not significantly different between groups (ET: 848 ± 155 mL·min⁻¹ and RA: 760 ± 131 mL·min⁻¹, P > 0.05). During the recovery period, the PCr offset time constant was significantly faster in ET compared with RA (ET: 32 ± 8 s and RA: 43 ± 10 s, P < 0.05), thus indicating an increased mitochondrial capacity for ATP synthesis in the quadriceps of ET. In contrast, the estimated mitochondrial efficiency during exercise was not significantly different (P/O, ET: 2.0 ± 1.0 and RA: 1.8 ± 0.4, P > 0.05). Consequently, the higher mitochondrial capacity for ATP synthesis in ET likely originated from an elevated mitochondrial volume density, mitochondria-specific respiratory capacity, and/or slower postexercise inactivation of oxidative phosphorylation by the parallel activation mechanism. CONCLUSION: Together, these findings reveal that 1) mitochondrial and contractile efficiencies are unaltered by several years of endurance training in young adults, and 2) the training-induced improvement in mitochondrial energetic capacity appears to be independent from changes in mitochondrial coupling.

Multi-atlas-based fully automatic segmentation of individual muscles in rat leg.

OBJECTIVE: To quantify individual muscle volume in rat leg MR images using a fully automatic multi-atlas-based segmentation method. MATERIALS AND METHODS: We optimized a multi-atlas-based segmentation method to take into account the voxel anisotropy of numbers of MRI acquisition protocols. We mainly tested an image upsampling process along Z and a constraint on the nonlinear deformation in the XY plane. We also evaluated a weighted vote procedure and an original implementation of an artificial atlas addition. Using this approach, we measured gastrocnemius and plantaris muscle volumes and compared the results with manual segmentation. The method reliability for volume quantification was evaluated using the relative overlap index. RESULTS: The most accurate segmentation was obtained using a nonlinear registration constrained in the XY plane by zeroing the Z component of the displacement and a weighted vote procedure for both muscles regardless of the number of atlases. The performance of the automatic segmentation and the corresponding volume quantification outperformed the interoperator variability using a minimum of three original atlases. CONCLUSION: We demonstrated the reliability of a multi-atlas segmentation approach for the automatic segmentation and volume quantification of individual muscles in rat leg and found that constraining the registration in plane significantly improved the results.

Opposite effects of hyperoxia on mitochondrial and contractile efficiency in human quadriceps muscles.

Exercise efficiency is an important determinant of exercise capacity. However, little is known about the physiological factors that can modulate muscle efficiency during exercise. We examined whether improved O2 availability would 1) impair mitochondrial efficiency and shift the energy production toward aerobic ATP synthesis and 2) reduce the ATP cost of dynamic contraction owing to an improved neuromuscular efficiency, such that 3) whole body O2 cost would remain unchanged. We used (31)P-magnetic resonance spectroscopy, surface electromyography, and pulmonary O2 consumption (V̇o2p) measurements in eight active subjects during 6 min of dynamic knee-extension exercise under different fractions of inspired O2 (FiO2 , 0.21 in normoxia and 1.0 in hyperoxia). V̇o2p (755 ± 111 ml/min in normoxia and 799 ± 188 ml/min in hyperoxia, P > 0.05) and O2 cost (P > 0.05) were not significantly different between normoxia and hyperoxia. In contrast, the total ATP synthesis rate and the ATP cost of dynamic contraction were significantly lower in hyperoxia than normoxia (P < 0.05). As a result, the ratio of the rate of oxidative ATP synthesis from the quadriceps to V̇o2p was lower in hyperoxia than normoxia but did not reach statistical significance (16 ± 3 mM/ml in normoxia and 12 ± 5 mM/ml in hyperoxia, P = 0.07). Together, these findings reveal dynamic and independent regulations of mitochondrial and contractile efficiency as a consequence of O2 availability in young active individuals. Furthermore, muscle efficiency appears to be already optimized in normoxia and is unlikely to contribute to the well-established improvement in exercise capacity induced by hyperoxia.

Fast imaging strategies for mouse kidney perfusion measurement with pseudocontinuous arterial spin labeling (pCASL) at ultra high magnetic field (11.75 tesla).

BACKGROUND: To derive an adapted protocol at ultra high magnetic field for mouse kidney perfusion measurements using pCASL in combination with three widely available fast imaging readouts: segmented SE EPI (sSE EPI), RARE, and TrueFISP. METHODS: pCASL sSE EPI, pCASL RARE, and pCASL TrueFISP were used for the acquisition of mouse kidney perfusion images in the axial and coronal planes at 11.75T. Results were compared in terms of perfusion sensitivity, signal-to-noise ratio (SNR), blood flow values, intrasession and intersession repeatability, and image quality (subjectively classified into three grades: good, satisfactory, and unacceptable). RESULTS: Renal cortex perfusion measurements were performed within 2 min with pCASL RARE/pCASL TrueFISP and 4 min with pCASL sSE EPI. In an axial direction, SNR values of 6.6/5.6/2.8, perfusion sensitivity values of 16.1 ± 3.7/13.6 ± 2.4/13.4 ± 1.0 %, blood flow values of 679 ± 149/466 ± 111/572 ± 46 mL/100 g/min and in-ROI variations values of 192/161/181 mL/100 g/min were obtained with pCASL sSE EPI/pCASL RARE/pCASL TrueFISP. Highest SNR per unit of time (1.8) and highest intra/intersession reliability (92.9% and 95.1%) were obtained with pCASL RARE, which additionally presented highly reproducible satisfactory image quality. In coronal plane, significantly lower SNR, perfusion sensitivity and perfusion values were obtained for all techniques compared with that in the axial plane (P < 0.05) due to magnetization saturation effects. CONCLUSION: pCASL RARE demonstrated more advantages for longitudinal preclinical kidney perfusion studies at ultra high magnetic field.

Localized metabolic and t2 changes induced by voluntary and evoked contractions.

PURPOSE: This study compared the metabolic and activation changes induced by electrically evoked (neuromuscular electrical stimulation (NMES)) and voluntary (VOL) contractions performed at the same submaximal intensity using P chemical shift imaging (CSI) and T2 mapping investigations. METHODS: Fifteen healthy subjects were asked to perform both NMES and VOL protocols with the knee extensors (i.e., 232 isometric contractions at 30% of maximal force) inside a 3-T scanner for two experimental sessions. During the first session, metabolic variations, i.e., phosphocreatine (PCr), inorganic phosphate (Pi), and pH, were recorded using localized P CSI. During a second session, T2 maps of the knee extensors were obtained at rest and immediately after each exercise. Voxels of interest were selected from the directly stimulated vastus lateralis and from the nondirectly stimulated rectus femoris/vastus intermedius muscles. RESULTS: PCr depletion recorded throughout the NMES session was significantly larger in the vastus lateralis as compared with the rectus femoris/vastus intermedius muscles for both conditions (VOL and NMES). A higher occurrence of Pi splitting and a greater acidosis was found during NMES as compared with VOL exercise, illustrating the heterogeneous activation of both slow and fast muscle fibers. T2 changes were greater after NMES as compared with VOL for both muscles but were not necessarily related to the localized metabolic demand. CONCLUSION: We provided direct evidence that the metabolic demand was strongly related to both the exercise modality and the site of stimulation. On the basis of the occurrence of Pi splitting, we suggested that NMES can activate fast muscle fibers even at low force levels.

Whole-brain quantitative mapping of metabolites using short echo three-dimensional proton MRSI.

BACKGROUND: To improve the extent over which whole brain quantitative three-dimensional (3D) magnetic resonance spectroscopic imaging (MRSI) maps can be obtained and be used to explore brain metabolism in a population of healthy volunteers. METHODS: Two short echo time (20 ms) acquisitions of 3D echo planar spectroscopic imaging at two orientations, one in the anterior commissure-posterior commissure (AC-PC) plane and the second tilted in the AC-PC +15° plane were obtained at 3 Tesla in a group of 10 healthy volunteers. B1 (+) , B1 (-) , and B0 correction procedures and normalization of metabolite signals with quantitative water proton density measurements were performed. A combination of the two spatially normalized 3D-MRSI, using a weighted mean based on the pixel wise standard deviation metabolic maps of each orientation obtained from the whole group, provided metabolite maps for each subject allowing regional metabolic profiles of all parcels of the automated anatomical labeling (AAL) atlas to be obtained. RESULTS: The combined metabolite maps derived from the two acquisitions reduced the regional intersubject variance. The numbers of AAL regions showing N-acetyl aspartate (NAA) SD/Mean ratios lower than 30% increased from 17 in the AC-PC orientation and 41 in the AC-PC+15° orientation, to a value of 76 regions of 116 for the combined NAA maps. Quantitatively, regional differences in absolute metabolite concentrations (mM) over the whole brain were depicted such as in the GM of frontal lobes (cNAA = 10.03 + 1.71; cCho = 1.78 ± 0.55; cCr = 7.29 ± 1.69; cmIns = 5.30 ± 2.67) and in cerebellum (cNAA = 5.28 ± 1.77; cCho = 1.60 ± 0.41; cCr = 6.95 ± 2.15; cmIns = 3.60 ± 0.74). CONCLUSION: A double-angulation acquisition enables improved metabolic characterization over a wide volume of the brain.

Fast water concentration mapping to normalize (1)H MR spectroscopic imaging.

OBJECT: To propose a fast and robust acquisition and post-processing pipeline that is time-compatible with clinical explorations to obtain a proton density (ρ) map used as a reference for metabolic map normalization. This allows inter-subject and inter-group comparisons of magnetic resonance spectroscopic imaging (MRSI) data and longitudinal follow-up for single subjects. MATERIALS AND METHODS: A multi-echo T 2 (*) mapping sequence, the XEP sequence for B 1 (+) -mapping and Driven Equilibrium Single Pulse Observation of T 1-an optimized variable flip angle method for T 1 mapping used for both B 1 (-) -mapping and M 0 calculation-were used to determine correction factors leading to quantitative water proton density maps at 3T. Normalized metabolite maps were obtained on a phantom and nine healthy volunteers. To show the potential use of this technique at the individual level, we also explored one patient with low-grade glioma. RESULTS: Accurate ρ maps were obtained both on phantom and volunteers. After signal normalization with the generated ρ maps, metabolic concentrations determined by the present method differed from theory by <7 % in the phantom and were in agreement with data from the literature for the healthy controls. Using these normalized metabolic values, it was possible to demonstrate in the patient with brain glioma, metabolic abnormalities in normalized N-acetyl aspartate, choline and creatine levels; illustrating the potential for direct use of this technique in clinical studies. CONCLUSION: The proposed combination of sequences provides a robust ρ map that can be used to normalize metabolic maps in clinical MRSI studies.

Hemi-spectrum substitution after water signal fitting (HESWAF): an improvement of the modulus post-processing of MR spectra.

OBJECTIVE: In a previous study, we have shown that modulus post-processing is a simple and efficient tool to both phase correct and frequency align magnetic resonance (MR) spectra automatically. Furthermore, this technique also eliminates sidebands and phase distortions. The advantages of the modulus technique have been illustrated in several applications to brain proton MR spectroscopy. Two possible drawbacks have also been pointed out. The first one is the theoretical decrease in signal-to-noise ratio (SNR) by a factor up to √2 when comparing the spectrum obtained after modulus versus conventional post-processing. The second pitfall results from the symmetrization of the spectrum induced by modulus post-processing, since any resonance or artifact located at the left of the water resonance is duplicated at the right of the water resonance, thus contaminating the region of the spectrum containing the resonances of interest. Herein, we propose a strategy in order to eliminate these two limitations. MATERIALS AND METHODS: Concerning the SNR issue, two complementary approaches are presented here. The first is based on the application of modulus post-processing before spatial apodization, and the second consists in substituting the left half of the spectrum by the fit of the water resonance before applying modulus post-processing. The symmetrization induced by modulus post-processing then combines the right half of the original spectrum containing the resonances of interest with the left half of the water fit, free of noise and artifacts. Consequently, the SNR is improved when compared to modulus post-processing alone. As a bonus, any artifact or resonance present in the left half of the original spectrum is removed. This solves the second limitation. RESULTS: After validation of the technique on simulations, we demonstrated that this improvement of the modulus technique is significantly advantageous for both in vitro and in vivo applications. CONCLUSION: By improving the SNR of the spectra and eliminating eventual contaminations, the new strategies proposed here confer an additional competitive advantage to the modulus post-processing technique.

Magnetization transfer from inhomogeneously broadened lines (ihMT): Experimental optimization of saturation parameters for human brain imaging at 1.5 Tesla.

PURPOSE: Recently a new MR endogenous contrast mechanism was reported. It allows specifically imaging the magnetization transfer (MT) effect arising from inhomogeneously broadened components of the NMR spectrum, and was hence dubbed ihMT. Such unique NMR lineshape properties are presumably occurring in myelin because of its specifically ordered, multilayered sheath structure. Here, optimization of a pulsed ihMT preparation module is presented to provide guidance for future studies and improve the understanding of underlying contrast mechanisms. METHODS: This study was performed at 1.5 Tesla on healthy volunteers. A pulsed ihMT preparation was implemented in combination with a HASTE readout module. The pulse width, interpulse repetition time, total saturation duration and RF saturation power were considered for optimization of the ihMT sensitivity and contrast. RESULTS: An optimal configuration of the preparation module was derived, leading to 10% ihMT signal in internal capsule (relative to unsaturated data) and around 200% signal increase relative to gray matter, i.e., approximately 10-fold superior contrast compared with conventional MT ratios, measured under similar experimental conditions. CONCLUSION: Overall the ihMT sequence was robust, sensitive and very specific for white matter. These findings suggest great potential for assessing brain myelination and for better characterization of myelin related disorders.

Metabolomic analysis of rat brain by high resolution nuclear magnetic resonance spectroscopy of tissue extracts.

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics and proteomics have been complemented by comprehensive quantitative analysis of the metabolite pool present in biological systems. This strategy, termed metabolomics, strives to provide a global characterization of the small-molecule complement involved in metabolism. While the genome and the proteome define the tasks cells can perform, the metabolome is part of the actual phenotype. Among the methods currently used in metabolomics, spectroscopic techniques are of special interest because they allow one to simultaneously analyze a large number of metabolites without prior selection for specific biochemical pathways, thus enabling a broad unbiased approach. Here, an optimized experimental protocol for metabolomic analysis by high-resolution NMR spectroscopy is presented, which is the method of choice for efficient quantification of tissue metabolites. Important strengths of this method are (i) the use of crude extracts, without the need to purify the sample and/or separate metabolites; (ii) the intrinsically quantitative nature of NMR, permitting quantitation of all metabolites represented by an NMR spectrum with one reference compound only; and (iii) the nondestructive nature of NMR enabling repeated use of the same sample for multiple measurements. The dynamic range of metabolite concentrations that can be covered is considerable due to the linear response of NMR signals, although metabolites occurring at extremely low concentrations may be difficult to detect. For the least abundant compounds, the highly sensitive mass spectrometry method may be advantageous although this technique requires more intricate sample preparation and quantification procedures than NMR spectroscopy. We present here an NMR protocol adjusted to rat brain analysis; however, the same protocol can be applied to other tissues with minor modifications.

Alterations at the cross-bridge level are associated with a paradoxical gain of muscle function in vivo in a mouse model of nemaline myopathy.

Nemaline myopathy is the most common disease entity among non-dystrophic skeletal muscle congenital diseases. The first disease causing mutation (Met9Arg) was identified in the gene encoding α-tropomyosin slow gene (TPM3). Considering the conflicting findings of the previous studies on the transgenic (Tg) mice carrying the TPM3Met9Arg mutation, we investigated carefully the effect of the Met9Arg mutation in 8-9 month-old Tg(TPM3)Met9Arg mice on muscle function using a multiscale methodological approach including skinned muscle fibers analysis and in vivo investigations by magnetic resonance imaging and 31-phosphorus magnetic resonance spectroscopy. While in vitro maximal force production was reduced in Tg(TPM3)Met9Arg mice as compared to controls, in vivo measurements revealed an improved mechanical performance in the transgenic mice as compared to the former. The reduced in vitro muscle force might be related to alterations occurring at the cross-bridges level with muscle-specific underlying mechanisms. In vivo muscle improvement was not associated with any changes in either muscle volume or energy metabolism. Our findings indicate that TPM3(Met9Arg) mutation leads to a mild muscle weakness in vitro related to an alteration at the cross-bridges level and a paradoxical gain of muscle function in vivo. These results clearly point out that in vitro alterations are muscle-dependent and do not necessarily translate into similar changes in vivo.

High-field (11.75T) multimodal MR imaging of exercising hindlimb mouse muscles using a non-invasive combined stimulation and force measurement device.

We have designed and constructed an experimental set-up allowing electrical stimulation of hindlimb mouse muscles and the corresponding force measurements at high-field (11.75T). We performed high-resolution multimodal MRI (including T2 -weighted imaging, angiography and diffusion) and analysed the corresponding MRI changes in response to a stimulation protocol. Mice were tested twice over a 1-week period to investigate the reliability of mechanical measurements and T2 changes associated with the stimulation protocol. Additionally, angiographic images were obtained before and immediately after the stimulation protocol. Finally, multislice diffusion imaging was performed before, during and immediately after the stimulation session. Apparent diffusion coefficient (ADC) maps were calculated on the basis of diffusion weighted images (DWI). Both force production and T2 values were highly reproducible as illustrated by the low coefficient of variation (<8%) and high intraclass correlation coefficient (≥0.75) values. Maximum intensity projection angiographic images clearly showed a strong vascular effect resulting from the stimulation protocol. Although a motion sensitive imaging sequence was used (echo planar imaging) and in spite of the strong muscle contractions, motion artifacts were minimal for DWI recorded under exercising conditions, thereby underlining the robustness of the measurements. Mean ADC values increased under exercising conditions and were higher during the recovery period as compared with the corresponding control values. The proposed experimental approach demonstrates accurate high-field multimodal MRI muscle investigations at a preclinical level which is of interest for monitoring the severity and/or the progression of neuromuscular diseases but also for assessing the efficacy of potential therapeutic interventions.

Effect of isoproterenol on myocardial perfusion, function, energy metabolism and nitric oxide pathway in the rat heart - a longitudinal MR study.

The chronic administration of the ß-adrenoreceptor agonist isoproterenol (IsoP) is used in animals to study the mechanisms of cardiac hypertrophy and failure associated with a sustained increase in circulating catecholamines. Time-dependent changes in myocardial blood flow (MBF), morphological and functional parameters were assessed in rats in vivo using multimodal cardiac MRI. Energy metabolism, oxidative stress and the nitric oxide (NO) pathway were evaluated in isolated perfused rat hearts following 7 days of treatment. Male Wistar rats were infused for 7 days with IsoP or vehicle using osmotic pumps. Cine-MRI and arterial spin labeling were used to determine left ventricular morphology, function and MBF at days 1, 2 and 7 after pump implantation. Isolated hearts were then perfused, and high-energy phosphate compounds and intracellular pH were followed using ³¹P MRS with simultaneous measurement of contractile function. Total creatine and malondialdehyde (MDA) contents were measured by high-performance liquid chromatography. The NO pathway was evaluated by NO synthase isoform expression and total nitrate concentration (NO(x)). In IsoP-treated rats, left ventricular mass was increased at day 1 and maintained. Wall thickness was increased with a peak at day 2 and a tendency to return to baseline values at day 7. MBF was markedly increased at day 1 and returned to normal values between days 1 and 2. The rate-pressure product and phosphocreatine/adenosine triphosphate ratio in perfused hearts were reduced. MDA, endothelial NO synthase expression and NO(x) were increased. Sustained high cardiac function and normal MBF after 24 h of IsoP infusion indicate imbalance between functional demand and blood flow, leading to morphological changes. After 1 week, cardiac hypertrophy and decreased function were associated with impaired phosphocreatine, increased oxidative stress and up-regulation of the NO pathway. These results provide supplemental information on the evolution of the different contributing factors leading to morphological and functional changes in this model of cardiac hypertrophy and failure.