Multiparametric Aging Study Across Adulthood in the Leg Through Quantitative MR Imaging, 1H Spectroscopy, and 31P Spectroscopy at 3T.

BACKGROUND: Improved characterization of healthy muscle aging is needed to establish early biomarkers in age-related diseases. PURPOSE: To quantify age-related changes on multiple MRI and clinical variables evaluated in the same cohort and identify correlations among them. STUDY TYPE: Prospective. POPULATION: 70 healthy subjects (30 men) from 20 to 81 years old. FIELD STRENGTH/SEQUENCE: 3T/water T2 (multiecho SE, multi-TE STEAM), water T1 (GRE MR Fingerprinting), fat-fraction (multiecho GRE, multi-TE STEAM), carnosine (PRESS), multicomponent water T2 (ISIS-CPMG SE train), and 31P pulse-acquire spectroscopy. ASSESSMENT: Age- and sex-related changes on: Imaging: fat-fraction (FFMRI), water T1 (T1-H2O), and T2 (T2-H2O-MRI) and their heterogeneities ΔT1-H2O and ΔT2-H2O-MRI in the posterior compartment (PC) and anterior compartment (AC) of the leg. 1H spectroscopy: Carnosine concentration, pH, water T2 components (T2-H2O-CPMG), fat-fraction (FFMRS), and water T2 (T2-H2O-MRS) in the gastrocnemius medialis. 31P spectroscopy: Phosphodiesters (PDE), phosphomonoesters, inorganic phosphates (Pi), and phosphocreatine (PCr) normalized to adenosine triphosphate (ATP) and pH in the calf. Clinical evaluation: Body-mass index (BMI), gait speed (GS), plantar flexion strength, handgrip strength (HS), HS normalized to wrist circumference (HSnorm), physical activity assessment. STATISTICAL TESTS: Multilinear regressions with sex and age as fixed factors. Spearman correlations calculated between variables. Benjamini-Hochberg procedure for false positives reduction (5% rate). A P < 0.05 significance level was used. RESULTS: Significant age-related increases were found for BMI (ρAge = 0.04), HSnorm (ρAge = -0.01), PDE/ATP (ρAge = 2.8 × 10-3), Pi/ATP (ρAge = 2.0 × 10-3), Pi/PCr (ρAge = 0.3 × 10-3), T2-H2O-MRS (ρAge = 0.051 msec), FFMRS (ρAge = 0.036) the intermediate T2-H2O-CPMG component time (ρAge = 0.112 msec), and fraction (ρAge = -0.3 × 10-3); and in both compartments for FFMRI (ρAge = 0.06, PC; ρAge = 0.06, AC), T2-H2O-MRI (ρAge = 0.05, PC; ρAge = 0.05, AC; msec), ΔT2-H2O-MRI (ρAge = 0.02, PC; ρAge = 0.02, AC; msec), T1-H2O (ρAge = 1.08, PC; ρAge = 1.06, AC; msec), and ΔT1-H2O (ρAge = 0.22, PC; ρAge = 0.37, AC; msec). The best age predictors, accounting for sex-related differences, were HSnorm (R2 = 0.52) and PDE/ATP (R2 = 0.44). In both leg compartments, the imaging measures and HSnorm were intercorrelated. In PC, T2-H2O-MRS and FFMRS also showed numerous correlations to the imaging measures. PDE/ATP correlated to T1-H2O, T2-H2O-MRI, ΔT2-H2O-MRI, FFMRI, FFMRS, the intermediate T2-H2O-CPMG, BMI, Pi/PCr, and HSnorm. DATA CONCLUSION: Our multiparametric MRI approach provided an integrative view of age-related changes in the leg and revealed multiple correlations between these parameters and the normalized HS. LEVEL OF EVIDENCE: 1 TECHNICAL EFFICACY: Stage 3.

Assessment of Extracellular Volume Fraction in Becker Muscular Dystrophy by Using MR Fingerprinting.

Background Quantitative MRI is increasingly proposed in clinical trials related to dystrophinopathies, including Becker muscular dystrophy (BMD). Purpose To establish the sensitivity of extracellular volume fraction (ECV) quantification using an MR fingerprinting sequence with water and fat separation as a quantitative imaging biomarker of skeletal muscle tissue alterations in BMD compared with fat fraction (FF) and water relaxation time quantification. Materials and Methods In this prospective study, study participants with BMD and healthy volunteers were included from April 2018 until October 2022 (ClinicalTrials.gov identifier NCT02020954). The MRI examination comprised FF mapping with the three-point Dixon method, water T2 mapping, and water T1 mapping before and after an intravenous injection of a gadolinium-based contrast agent by using MR fingerprinting, from which ECV was calculated. Functional status was measured with use of the Walton and Gardner-Medwin scale. This clinical evaluation tool stratifies disease severity from grade 0 (preclinical; elevated creatine phosphokinase; all activities normal) to grade 9 (unable to eat, drink, or sit without assistance). Mann-Whitney U tests, Kruskal-Wallis tests, and Spearman rank correlation tests were performed. Results Twenty-eight participants with BMD (median age, 42 years [IQR, 34-52 years]; 28 male) and 19 healthy volunteers (median age, 39 years [IQR, 33-55 years]; 19 male) were evaluated. ECV was higher in participants with dystrophy than in controls (median, 0.21 [IQR, 0.16-0.28] vs 0.07 [IQR, 0.07-0.08]; P < .001). In muscles of participants with BMD with normal FF, ECV was also higher than in muscles of healthy controls (median, 0.11 [IQR, 0.10-0.15] vs 0.07 [IQR, 0.07-0.08]; P = .02). ECV was correlated with FF (ρ = 0.56, P = .003), Walton and Gardner-Medwin scale score (ρ = 0.52, P = .006), and serum cardiac troponin T level (ρ = 0.60, P < .001). Conclusion Quantitative MR relaxometry with water and fat separation indicates a significant increase of skeletal muscle extracellular volume fraction in study participants with Becker muscular dystrophy. Clinical trial registration no. NCT02020954 Published under a CC BY 4.0 license. Supplemental material is available for this article.

Charcot-Marie-Tooth disease type 2CC due to NEFH variants causes a progressive, non-length-dependent, motor-predominant phenotype.

OBJECTIVE: Neurofilaments are the major scaffolding proteins for the neuronal cytoskeleton, and variants in NEFH have recently been described to cause axonal Charcot-Marie-Tooth disease type 2CC (CMT2CC). METHODS: In this large observational study, we present phenotype-genotype correlations on 30 affected and 3 asymptomatic mutation carriers from eight families. RESULTS: The majority of patients presented in adulthood with motor-predominant and lower limb-predominant symptoms and the average age of onset was 31.0±15.1 years. A prominent feature was the development of proximal weakness early in the course of the disease. The disease progressed rapidly, unlike other Charcot-Marie-Tooth disease (CMT) subtypes, and half of the patients (53%) needed to use a wheelchair on average 24.1 years after symptom onset. Furthermore, 40% of patients had evidence of early ankle plantarflexion weakness, a feature which is observed in only a handful of CMT subtypes. Neurophysiological studies and MRI of the lower limbs confirmed the presence of a non-length-dependent neuropathy in the majority of patients.All families harboured heterozygous frameshift variants in the last exon of NEFH, resulting in a reading frameshift to an alternate open reading frame and the translation of approximately 42 additional amino acids from the 3' untranslated region (3'-UTR). CONCLUSIONS: This phenotype-genotype study highlights the unusual phenotype of CMT2CC, which is more akin to spinal muscular atrophy rather than classic CMT. Furthermore, the study will enable more informative discussions on the natural history of the disease and will aid in NEFH variant interpretation in the context of the disease's unique molecular genetics.

Water-Fat Separation in MR Fingerprinting for Quantitative Monitoring of the Skeletal Muscle in Neuromuscular Disorders.

Background Quantitative MRI is increasingly proposed in clinical trials related to neuromuscular disorders (NMDs). Purpose To investigate the potential of an MR fingerprinting sequence for water and fat fraction (FF) quantification (MRF T1-FF) for providing markers of fatty replacement and disease activity in patients with NMDs and to establish the sensitivity of water T1 as a marker of disease activity compared with water T2 mapping. Materials and Methods Data acquired between March 2018 and March 2020 from the legs of patients with NMDs were retrospectively analyzed. The MRI examination comprised fat-suppressed T2-weighted imaging, mapping of the FF measured with the three-point Dixon technique (FFDixon), water T2 mapping, and MRF T1-FF, from which the FF measured with MRF T1-FF (FFMRF) and water T1 were derived. Data from the legs of healthy volunteers were prospectively acquired between January and July 2020 to derive abnormality thresholds for FF, water T2, and water T1 values. Kruskal-Wallis tests and receiver operating characteristic curve analysis were performed, and linear models were used. Results A total of 73 patients (mean age ± standard deviation, 47 years ± 12; 45 women) and 15 healthy volunteers (mean age, 33 years ± 8; three women) were evaluated. A linear correlation was observed between FFMRF and FFDixon (R2 = 0.97, P < .001). Water T1 values were higher in muscles with high signal intensity at fat-suppressed T2-weighted imaging than in muscles with low signal intensity (mean value, 1281 msec [95% CI: 1165, 1604] vs 1198 msec [95% CI: 1099, 1312], respectively; P < .001), and a correlation was found between water T1 and water T2 distribution metrics (R2 = 0.66 and 0.79 for the median and 90th percentile values, respectively; P < .001). Water T1 classified the patients' muscles as abnormal based on quantitative water T2, with high sensitivity (93%; 68 of 73 patients) and specificity (80%; 53 of 73 patients) (area under the receiver operating characteristic curve, 0.92 [95% CI: 0.83, 0.97]; P < .001). Conclusion Water-fat separation in MR fingerprinting is robust for deriving quantitative imaging markers of intramuscular fatty replacement and disease activity in patients with neuromuscular disorders. © RSNA, 2021 Online supplemental material is available for this article.

Heterozygous LmnadelK32 mice develop dilated cardiomyopathy through a combined pathomechanism of haploinsufficiency and peptide toxicity.

Dilated cardiomyopathy (DCM) associates left ventricular (LV) dilatation and systolic dysfunction and is a major cause of heart failure and cardiac transplantation. LMNA gene encodes lamins A/C, proteins of the nuclear envelope. LMNA mutations cause DCM with conduction and/or rhythm defects. The pathomechanisms linking mutations to DCM remain to be elucidated. We investigated the phenotype and associated pathomechanisms of heterozygous Lmna(ΔK32/+) (Het) knock-in mice, which carry a human mutation. Het mice developed a cardiac-specific phenotype. Two phases, with two different pathomechanisms, could be observed that lead to the development of cardiac dysfunction, DCM and death between 35 and 70 weeks of age. In young Het hearts, there was a clear reduction in lamin A/C level, mainly due to the degradation of toxic ΔK32-lamin. As a side effect, lamin A/C haploinsufficiency probably triggers the cardiac remodelling. In older hearts, when DCM has developed, the lamin A/C level was normalized and associated with increased toxic ΔK32-lamin expression. Crossing our mice with the Ub(G76V)-GFP ubiquitin-proteasome system (UPS) reporter mice revealed a heart-specific UPS impairment in Het. While UPS impairment itself has a clear deleterious effect on engineered heart tissue's force of contraction, it also leads to the nuclear aggregation of viral-mediated expression of ΔK32-lamin. In conclusion, Het mice are the first knock-in Lmna model with cardiac-specific phenotype at the heterozygous state. Altogether, our data provide evidence that Het cardiomyocytes have to deal with major dilemma: mutant lamin A/C degradation or normalization of lamin level to fight the deleterious effect of lamin haploinsufficiency, both leading to DCM.

Forelimb treatment in a large cohort of dystrophic dogs supports delivery of a recombinant AAV for exon skipping in Duchenne patients.

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disorder caused by mutations in the dystrophin gene, without curative treatment yet available. Our study provides, for the first time, the overall safety profile and therapeutic dose of a recombinant adeno-associated virus vector, serotype 8 (rAAV8) carrying a modified U7snRNA sequence promoting exon skipping to restore a functional in-frame dystrophin transcript, and injected by locoregional transvenous perfusion of the forelimb. Eighteen Golden Retriever Muscular Dystrophy (GRMD) dogs were exposed to increasing doses of GMP-manufactured vector. Treatment was well tolerated in all, and no acute nor delayed adverse effect, including systemic and immune toxicity was detected. There was a dose relationship for the amount of exon skipping with up to 80% of myofibers expressing dystrophin at the highest dose. Similarly, histological, nuclear magnetic resonance pathological indices and strength improvement responded in a dose-dependent manner. The systematic comparison of effects using different independent methods, allowed to define a minimum threshold of dystrophin expressing fibers (>33% for structural measures and >40% for strength) under which there was no clear-cut therapeutic effect. Altogether, these results support the concept of a phase 1/2 trial of locoregional delivery into upper limbs of nonambulatory DMD patients.

New insights on human skeletal muscle tissue compartments revealed by in vivo t2 NMR relaxometry.

The spin-spin (T2) relaxation of (1)H-NMR signals in human skeletal muscle has been previously hypothesized to reveal information about myowater compartmentation. Although experimental support has been provided, no consensus has yet emerged concerning the attribution of specific anatomical compartments to the observed T2 components. Potential application of a noninvasive tool that might offer such information urges the quest for a definitive answer to this question. The purpose of this work was to obtain new information that might help elucidate the mechanism of T2 distribution in muscle. To do so, in vivo T2 relaxation data was acquired from the soleus of eight healthy volunteers using a localized Carr-Purcell-Meiboom-Gill technique. Each acquisition contained 1000 echoes with an interecho spacing of 1 ms. Data were acquired from each subject under different vascular filling preparations expected to change exclusively the extracellular water fraction. Two exponential components were systematically observed: an intermediate component (T2 ~ 32 ms) and a long component (100 < T2 < 210 ms). The relative fraction and T2 value characterizing the long component systematically increased after progressive augmentation of extracellular water volume. Characteristic relaxation behavior for each vascular filling condition was analyzed with a two-site exchange model and a three-site two-exchange model. We show that a two-site exchange model can only predict the observations for small exchange rates, much more representative of transendothelial than transcytolemmal exchange regimes. The three-site two-exchange model representing the intracellular, interstitial, and vascular spaces was capable of precisely predicting the observations for realistic transcytolemmal and transendothelial exchange rates. The estimated intrinsic relative fractions of each of these compartments corroborate with estimations from previous works and strongly suggest that the T2 relaxation from water within the intracellular and interstitial spaces is described by the intermediate component, whereas the long component represents water within the vascular space.

Arginine butyrate per os protects mdx mice against cardiomyopathy, kyphosis and changes in axonal excitability.

Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disease caused by lack of dystrophin, a sub-sarcolemmal protein, which leads to dramatic muscle deterioration. We studied in mdx mice, the effects of oral administration of arginine butyrate (AB), a compound currently used for the treatment of sickle cell anemia in children, on cardiomyopathy, vertebral column deformation and electromyographic abnormalities. Monthly follow-up by echocardiography from the 8th month to the 14th month showed that AB treatment protected the mdx mice against drastic reduction (20-23%) of ejection fraction and fractional shortening, and also against the ≈20% ventricular dilatation and 25% cardiac hypertrophy observed in saline-treated mdx mice. The phenotypic improvement was corroborated by the decrease in serum CK level and by better fatigue resistance. Moreover, AB treatment protected against the progressive spinal deformity observed in mdx mice, another similarity with DMD patients. The value of the kyphosis index in AB-treated mice reached 94% of the value in C57BL/10 mice. Finally, axonal excitability parameters such as the membrane resting potential, the threshold and amplitude of the action potential, the absolute and relative refractory periods and the supernormal and subnormal periods, recorded from caudal and plantar muscles in response to excitability tests, that were modified in saline-treated mdx mice were not significantly changed, compared with wild-type animals, in AB-treated mdx mice. All of these results suggest that AB could be a potential treatment for DMD patients.

GDF15 mediates renal cell plasticity in response to potassium depletion in mice.

OBJECTIVE: To understand the mechanisms involved in the response to a low-K+ diet (LK), we investigated the role of the growth factor GDF15 and the ion pump H,K-ATPase type 2 (HKA2) in this process. METHODS: Male mice of different genotypes (WT, GDF15-KO, and HKA2-KO) were fed an LK diet for different periods of time. We analyzed GDF15 levels, metabolic and physiological parameters, and the cellular composition of collecting ducts. RESULTS: Mice fed an LK diet showed a 2-4-fold increase in plasma and urine GDF15 levels. Compared to WT mice, GDF15-KO mice rapidly developed hypokalemia due to impaired renal adaptation. This is related to their 1/ inability to increase the number of type A intercalated cells (AIC) and 2/ absence of upregulation of H,K-ATPase type 2 (HKA2), the two processes responsible for K+ retention. Interestingly, we showed that the GDF15-mediated proliferative effect on AIC was dependent on the ErbB2 receptor and required the presence of HKA2. Finally, renal leakage of K+ induced a reduction in muscle mass in GDF15-KO mice fed LK diet. CONCLUSIONS: In this study, we showed that GDF15 and HKA2 are linked and play a central role in the response to K+ restriction by orchestrating the modification of the cellular composition of the collecting duct.

Muscle MRI as a Diagnostic Challenge in Emery-Dreifuss Muscular Dystrophy.

 Emery-Dreifuss Muscular Dystrophy (EDMD) is an early-onset, slowly-progressive group of myopathies, presenting with joint contractures, muscle weakness and cardiac abnormalities. Variants in the EMD gene cause an X-linked recessive form (EDMD1). The scarce EDMD1 muscle MRI accounts in the literature describe fatty replacement of posterior thigh and leg muscles.We report a 22-year-old patient with early-onset bilateral joint contractures, slowly progressive muscle weakness and minor cardiac rhythm abnormalities. A novel loss-of-function variant of EMD was identified and deemed probably pathogenic in the absence of emerin detection by immunofluorescence and Western Blot. MRI revealed fatty replacement of the lumbar spinal erectors and the posterior compartment of lower limbs. Interestingly, Short Tau Inversion Recovery (STIR) sequences showed a heterogenous hyper signal on the vasti, hamstrings and left lateral gastrocnemius muscles.Oedema-like abnormalities were previously reported in early stages of other muscular dystrophies, preceding fatty replacement and muscle atrophy, but not in EDMD1 patients. We hypothesize that these oedema-like changes may be a marker of early muscle pathology in EDMD1. Further studies focusing on these abnormalities in the early phase of EDMD1 are required to test our hypothesis.

Non-invasive assessment of skeletal muscle fibrosis in mice using nuclear magnetic resonance imaging and ultrasound shear wave elastography.

Fibrosis is a key pathological feature in muscle disorders, but its quantification mainly relies on histological and biochemical assays. Muscle fibrosis most frequently is entangled with other pathological processes, as cell membrane lesions, inflammation, necrosis, regeneration, or fatty infiltration, making in vivo assessment difficult. Here, we (1) describe a novel mouse model with variable levels of induced skeletal muscle fibrosis displaying minimal inflammation and no fat infiltration, and (2) report how fibrosis affects non-invasive metrics derived from nuclear magnetic resonance (NMR) and ultrasound shear-wave elastography (SWE) associated with a passive biomechanical assay. Our findings show that collagen fraction correlates with multiple non-invasive metrics. Among them, muscle stiffness as measured by SWE, T2, and extracellular volume (ECV) as measured by NMR have the strongest correlations with histology. We also report that combining metrics in a multi-modality index allowed better discrimination between fibrotic and normal skeletal muscles. This study demonstrates that skeletal muscle fibrosis leads to alterations that can be assessed in vivo with multiple imaging parameters. Furthermore, combining NMR and SWE passive biomechanical assay improves the non-invasive evaluation of skeletal muscle fibrosis and may allow disentangling it from co-occurring pathological alterations in more complex scenarios, such as muscular dystrophies.

Ca2+ overload and mitochondrial permeability transition pore activation in living delta-sarcoglycan-deficient cardiomyocytes.

Muscular dystrophies are often associated with significant cardiac disease that can be the prominent feature associated with gene mutations in sarcoglycan. Cardiac cell death is a main feature of cardiomyopathy in sarcoglycan deficiency and may arise as a cardiomyocyte intrinsic process that remains unclear. Deficiency of delta-sarcoglycan (delta-SG) induces disruption of the dystrophin-associated glycoprotein complex, a known cause of membrane instability that may explain cardiomyocytes cytosolic Ca2+ increase. In this study we assessed the hypothesis that cytosolic Ca2+ increase triggers cardiomyocyte death through mitochondrial Ca2+ overload and dysfunction in the delta-SG-deficient CHF147 hamster. We showed that virtually all isolated CHF147 ventricular myocytes exhibited elevated cytosolic and mitochondrial Ca2+ levels by the use of the Fura-2 and Rhod-2 fluorescent probes. Observation of living cells with Mito-Tracker red lead to the conclusion that approximately 15% of isolated CHF147 cardiomyocytes had disorganized mitochondria. Transmission electron microscope imaging showed mitochondrial swelling associated with crest and membrane disruption. Analysis of the mitochondrial permeability transition pore (MPTP) activity using calcein revealed that mitochondria of CHF147 ventricular cells were twofold leakier than wild types, whereas reactive oxygen species production was unchanged. Bax, Bcl-2, and LC3 expression analysis by Western blot indicated that the intrinsic apoptosis and the cell death associated to autophagy pathways were not significantly activated in CHF147 hearts. Our results lead to conclusion that cardiomyocytes death in delta-SG-deficient animals is an intrinsic phenomenon, likely related to Ca2+-induced necrosis. In this process Ca2+ overload-induced MPTP activation and mitochondrial disorganization may have an important role.

Non-invasive and quantitative evaluation of peripheral vascular resistances in rats by combined NMR measurements of perfusion and blood pressure using ASL and dynamic angiography.

The in vivo determination of peripheral vascular resistances (VR) is crucial for the assessment of arteriolar function. It requires simultaneous determination of organ perfusion (F) and arterial blood pressure (BP). A fully non-invasive method was developed to measure systolic and diastolic BP in the caudal artery of rats based on dynamic NMR angiography. A good agreement was found between the NMR approach and the gold standard techniques (linear regression slope = 0.98, R(2) = 0.96). This method and the ASL-MRI measurement of skeletal muscle perfusion were combined into one single NMR experiment to quantitatively evaluate the local vascular resistances in the calf muscle of anaesthetized rats, in vivo and non-invasively 1) at rest: VR = 7.0 +/- 1.0 mmHg x min 100 g x ml(-1), F = 13 +/- 3 ml min(-1) x 100 g(-1) and mean BP (MBP) = 88 +/- 10 mmHg; 2) under vasodilator challenge (milrinone): VR = 3.7 +/- 1.1 mmHg min x 100 g ml(-1), F = 21 +/- 4 ml min(-1) x 100 g(-1) and MBP = 75 +/- 14 mmHg; 3) under vasopressor challenge (norepinephrine): VR = 9.8 +/- 1.2 mmHg min 100 g ml(-1), F = 14 +/- 3 ml min(-1) x 100 g(-1) and MBP = 137 +/- 2 mmHg.

Capsid-specific removal of circulating antibodies to adeno-associated virus vectors.

Neutralizing antibodies directed against adeno-associated virus (AAV) are commonly found in humans. In seropositive subjects, vector administration is not feasible as antibodies neutralize AAV vectors even at low titers. Consequently, a relatively large proportion of humans is excluded from enrollment in clinical trials and, similarly, vector redosing is not feasible because of development of high-titer antibodies following AAV vector administration. Plasmapheresis has been proposed as strategy to remove anti-AAV antibodies from the bloodstream. Although safe and relatively effective, the technology has some limitations mainly related to the nonspecific removal of all circulating IgG. Here we developed an AAV-specific plasmapheresis column which was shown to efficiently and selectively deplete anti-AAV antibodies without depleting the total immunoglobulin pool from plasma. We showed the nearly complete removal of anti-AAV antibodies from high titer purified human IgG pools and plasma samples, decreasing titers to levels that allow AAV vector administration in mice. These results provide proof-of-concept of a method for the AAV-specific depletion of neutralizing antibodies in the setting of in vivo gene transfer.

Comparison of toxicity and transfection efficiency of amphiphilic block copolymers and polycationic polymers in striated muscles.

BACKGROUND: Gene delivery using synthetic vectors is frequently based on cationic compounds such as polyethyleneimine (PEI). However, few data have been published on the ability of PEI to mediate transgene expression in muscle tissue. Besides cationic vectors, there is increasing interest focusing on amphiphilic copolymers as gene carriers into striated muscles, although their mechanism of action is unknown. METHODS: Plasmid DNA was associated with three different polymers: the cationic polyethyleneimine and two amphiphilic copolymers displaying few (tetronic 304) or no charges (pluronic L64). The resulting formulations were investigated by dynamic light scattering, laser doppler velocimetry, gel retardation assay and transmission electron microscopy. The toxicity and efficiency of the carriers were assessed in both skeletal and cardiac muscles. RESULTS: PEI efficiently condenses plasmids into small complexes displaying a positive electrophoretic mobility. However, these PEI/DNA complexes lead to severe side-effects in vivo. The association between amphiphilic copolymers and DNA leads to no or only partial condensation of plasmids. Moreover, amphiphilic polymers do not interact strongly with DNA and tetronic-based systems are destabilized with a decreasing pH. Those vectors also display a negative electrophoretic mobility. Thus, characteristics of amphiphilic polymer/DNA systems might be considered unfavourable for transfection. However, safe and rather efficient gene expression was obtained in skeletal muscles, even at low DNA doses, but not in the myocardium. CONCLUSIONS: The present study highlights the interest in amphiphilic carriers for promoting DNA transfection in vivo. Gaining new insights into the properties of these vectors should allow their optimization.

Maternal serotonin influences cardiac function in adult offspring.

Using the Tph1-invalidated mouse line, in which blood is depleted in serotonin (5-hydroxytryptamine, 5-HT), we have demonstrated previously that maternal 5-HT is required for normal embryonic development. Here, we address the issue of the influence of the maternal 5-HT concentration on the cardiac function of the offspring as adults. We investigated the cardiac phenotype of Tph1-invalidated mice born to Tph1 heterozygous and null mothers. Functionally, all mutants display a significant decrease of cardiac contractility, indicative of impaired left ventricular function. They exhibit progressive dilated cardiomyopathy and are unable to adapt appropriately to a pharmacological stress. Moreover, we show that the cardiopathy is more severe in adult Tph1(-/-) mice born to homozygous mothers than to heterozygous mothers. Importantly, the severity of the cardiac phenotype is inversely correlated with the plasma 5-HT concentration but not the whole-blood 5-HT concentration. Thus, plasma 5-HT concentration may be a useful index of heart failure. These findings show that cardiac function, through the plasma 5-HT concentration, is influenced by the maternal serotonergic status.

MIECT: How did it start?

Transiently assuming the functions of both heart and lungs as surgeons repair critical valves and vessel lesions can be achieved by mechanical circulatory support has its origins in cardiopulmonary bypass (CPB). However, CPB technologies induce also some unintended adverse effects. During the 90s, a mayor trend pushed many physicians to reconsider the place of coronary artery bypass grafting (CABG) and challenged the surgical reference treatment by less invasive catheter-based angioplasties. Nevertheless, best long-term patient outcomes were related to surgery. Therefore, a small number of multidisciplinary teams in Regensburg and Paris started to develop a minimally invasive CPB system. The basic concept relied on a closed-loop perfusion circuit with a non-occlusive pump. Moreover, the team in Paris pushed the concept further and developed a complete fully integrated CPB system allowed first closed-heart and later open-heart surgery with aortic cross-clamping and efficient cardioprotection. Those were the initial steps towards the future developments of minimally invasive extracorporeal circulation technologies. Initial clinical results were clearly positive in terms of overall morbimortality. Moreover, several preliminary results pointed out the biological benefits that decreased hemodilution, improved preservation of the immune reactions and more stable anticoagulation could bring to the field of ECT.

Quantitative, dynamic and noninvasive determination of skeletal muscle perfusion in mouse leg by NMR arterial spin-labeled imaging.

Because mouse may relatively easily be genetically tailored to develop equivalent of human muscular diseases or to present controlled alterations of mechanisms involved in vasoregulation, it has become the prevalent species to explore such questions. However, the very small size of the animals represents a serious limitation when evaluating the functional consequences of these genetic manipulations. In this context, the recourse to arterial spin labeling (ASL) nuclear magnetic resonance (NMR) methods in which arterial water spins act as an endogenous and freely diffusible tracer of perfusion is tempting but challenging. This article shows that despite the small size of the animal, mouse muscle perfusion may be measured, at rest and in conditions of reactive hyperemia, using saturation inversion recovery sequence, a pulsed ASL variant, combined with NMR imaging. Baseline perfusion values in the mouse leg were 17+/-11 ml.min(-1).100 g(-1) (n=11) and were comparable to microsphere data from the literature. Under ischemia, leg perfusion was 1.2+/-9.3 ml.min(-1).100 g(-1) (n=11). The difference observed between basal and ischemic measurements was statistically different (P=.0001). The temporal pattern of hyperemia in mouse muscle was coherent with previously published measurements in humans and in rats. The mean peak perfusion was 62+/-24 ml.min(-1).100 g(-1) (n=6) occurring 48+/-27 s after the end of occlusion. In conclusion, this study demonstrated the ability of ASL combined to NMR imaging to quantify skeletal muscle perfusion in mice legs, both at rest and dynamically.

Eight-year results of Freestyle stentless bioprosthesis in the aortic position: a single-center study of 500 patients.

BACKGROUND AND AIM OF THE STUDY: Stentless bioprostheses may be the future valve of choice for aortic valve replacement (AVR). The study aim was to investigate mid-term clinical outcome after AVR with the Medtronic Freestyle valve. METHODS: Between April 1997 and November 2004, a total of 500 patients (241 females, 259 males) was implanted with a Freestyle bioprosthesis for AVR, without population selection, by a single surgical team at the authors' institutions. Mean patient age was 74.5 +/- 9.6 years (range: 26-91 years); 34 patients (7%) were aged < 60 years, 121 (24%) were aged > 80 years, and 205 (41%) were in NYHA classes III or IV. The surgical procedure used included a modified subcoronary technique in 482 cases and complete root replacement in 18, conducted with mini-extracorporeal circulation. Concomitant procedures included coronary artery bypass grafting in 123 patients (25%), mitral valve repair/replacement in five, and maze in two. Follow up was 98% complete; the mean follow up was 31.3 months (range: 4-95 months). RESULTS: The mean cardiopulmonary bypass time was 98 +/- 26 min, and total aortic cross-clamp time 77 +/- 19 min. Operative mortality was 5.2% (n = 26), and no patients aged under 60 years died. At eight years, freedom from structural valve deterioration was 100% (0% in the young population), freedom from endocarditis 97.2%, freedom from reoperation 97%, and overall survival 83%. Most of the late deaths (n = 56) were of non-cardiac origin, and occurred in older patients. After one year, the mean aortic echocardiographic gradient was 11.5 +/- 1.1 mmHg, and was improved compared to that at discharge. No significant aortic insufficiency occurred. CONCLUSION: Use of the Freestyle stentless bioprosthesis for AVR resulted in excellent short-term survival in the octogenarian population, and excellent mid-term results in the younger population. In time, experience will indicate whether the Freestyle should be considered as the bioprosthesis of choice for patients of all ages.