A feasability study of color flow doppler vectorization for automated blood flow monitoring.

An ongoing issue in vascular medicine is the measure of the blood flow. Catheterization remains the gold standard measurement method, although non-invasive techniques are an area of intense research. We hereby present a computational method for real-time measurement of the blood flow from color flow Doppler data, with a focus on simplicity and monitoring instead of diagnostics. We then analyze the performance of a proof-of-principle software implementation. We imagined a geometrical model geared towards blood flow computation from a color flow Doppler signal, and we developed a software implementation requiring only a standard diagnostic ultrasound device. Detection performance was evaluated by computing flow and its determinants (flow speed, vessel area, and ultrasound beam angle of incidence) on purposely designed synthetic and phantom-based arterial flow simulations. Flow was appropriately detected in all cases. Errors on synthetic images ranged from nonexistent to substantial depending on experimental conditions. Mean errors on measurements from our phantom flow simulation ranged from 1.2 to 40.2% for angle estimation, and from 3.2 to 25.3% for real-time flow estimation. This study is a proof of concept showing that accurate measurement can be done from automated color flow Doppler signal extraction, providing the industry the opportunity for further optimization using raw ultrasound data.

Placental BDNF/TrkB signaling system is modulated by fetal growth disturbances in rat and human.

The brain-derived neurotrophic factor (BDNF) has been shown to exert an important role during implantation, placental development, and fetal growth control in mice. Its expression is closely related to the nutritional status in several tissues such as in the nervous system. In a previous study, we demonstrated that maternal undernutrition (MU), during the perinatal life, modified both the BDNF and its functional receptor, the tyrosine kinase receptor B (TrkB) gene expression in the brain of growth-restricted rat offspring during sensitive developmental windows, suggesting that these early modifications may have long-lasting consequences. In the present study, we measured BDNF/TrkB mRNA and protein levels in rat placentas from mothers submitted to a 50% food restriction during gestation, and in human placentas from pregnancies with fetal growth restriction or fetal macrosomia. In the rat, two subtypes of placental TrkB receptors have been identified: the TrkB-FL and TrkB-T1 receptors. We found that MU induced intrauterine growth restriction (IUGR) of fetuses at term and decreased the placental BDNF mRNA and protein levels. Placentae from undernourished mothers exhibited an increased mRNA expression of TrkB-FL whereas both TrkB-FL and TrkB-T1 receptors proteins levels were not modified. In human IUGR placentas, both BDNF and TrkB receptor mRNA expressions were up-regulated. Finally, although neither BDNF nor TrkB mRNA levels were altered by fetal macrosomia alone, BDNF mRNA levels were decreased when macrosomia was associated with maternal type 1 diabetes. These results show that the placental BDNF/TrkB system is modulated in rats and humans during pregnancies with fetal growth perturbations and is affected by the maternal energetic status. These data suggest that this system may exert an important role for the feto-placental unit development and that it may also be implicated in the etiology of pathologies related to placental and fetal growth disturbances.

O-GlcNAcylation, an original modulator of contractile activity in striated muscle.

There is growing evidence that O-linked N-acetyl-D-glucosaminylation, more simply termed O-GlcNAcylation or O-GlcNAc, is a post-translational modification involved in many cellular processes from transcription to modulation of protein properties. O-GlcNAc is a dynamic and reversible glycosylation and therefore quite similar to the phosphorylation/dephosphorylation process, with which O-GlcNAc can interplay. Since O-GlcNAc serves as a glucose sensor by the way of hexosamine biosynthesis pathway, this glycosylation is often associated with glucose toxicity and development of insulin resistance. In this way, O-GlcNAc could be involved in muscle pathological consequences of diabetes. Nevertheless, in regards of several studies performed in healthy striated muscles, O-GlcNAc seems to exert protective effects against different types of injuries. Recent new insights suggest a key implication of O-GlcNAc in skeletal and cardiac muscles contractile activity, in particular by O-GlcNAc modification of motor as well as regulating contractile proteins. While evidence linked O-GlcNAc to the regulation of calcium activation properties, its exact role remains to be defined as well as the existence of potential interference with phosphorylation. The better understanding of the exact function of OGlcNAc in this physiological process could contribute to the determination of newly markers of skeletal dysfunctions.

Childhood spinal muscular atrophy induces alterations in contractile and regulatory protein isoform expressions.

AIMS: Although modifications of the survival motor neurone gene are responsible for most spinal muscular atrophy (SMA) cases, the molecular pathophysiology and the muscular target proteins involved are still unknown. The aim of this study was to compare the expression of contractile and regulatory protein isoforms in quadriceps muscles from SMA children with age-matched control quadriceps. METHODS: The isoform patterns of myosin heavy chains (MHC), troponin subunits (T, C and I) and tropomyosin were determined by immunoblotting, reverse transcription-polymerase chain reaction and mass spectrometry analyses. Depending on the disease severity, their expression levels were followed in specific variants of SMA populations (types I, II and III), with comparison with age-matched control muscles. RESULTS: The isoform transitions in SMA muscles were different from the fast-to-faster transitions occurring in normal muscles from children aged 1 month to 5 years old. Moreover, the expression of the neonatal MHC isoform was not repressed in SMA muscles. CONCLUSIONS: The presence of the neonatal MHC isoform in SMA muscles indicates an alteration of the phenotype in these diseased muscles. It is strongly suggested that MHC and troponin T proteins may be good markers for the SMA pathology.

Dual effect of deafferentation on contractile characteristics and sarcoplasmic reticulum properties in rat soleus fibers.

The neural message is known to play a key role in muscle development and function. We analyzed the specific role of the afferent message on the functional regulation of two subcellular muscle components involved in the contractile mechanism: the contractile proteins and the sarcoplasmic reticulum (SR). Rats were submitted to bilateral deafferentation (DEAF group) by section of the dorsal roots L(3) to L(5) after laminectomy. Experiments were carried out in single skinned fibers of the soleus muscle. The maximal force developed by the contractile proteins was increased in the DEAF group compared with control, despite a decrease in muscle mass by 17%. The tension-pCa relationship was shifted toward lower calcium (Ca(2+)) concentrations. Different functional properties of the SR of DEAF soleus were examined by using caffeine-induced contractions. The caffeine sensitivity of the Ca(2+) release was decreased after deafferentation and ryanodine receptor 1 isoform was expressed at a lower level. The rate of Ca(2+) uptake was only slightly increased. The results underlined the dual effect of the afferent input on the functional regulation of both contractile proteins and SR.

Expression and functional properties of four slow skeletal troponin T isoforms in rat muscles.

We investigated the expression and functional properties of slow skeletal troponin T (sTnT) isoforms in rat skeletal muscles. Four sTnT cDNAs were cloned from the slow soleus muscle. Three isoforms were found to be similar to sTnT1, sTnT2, and sTnT3 isoforms described in mouse muscles. A new rat isoform, with a molecular weight slightly higher than that of sTnT3, was discovered. This fourth isoform had never been detected previously in any skeletal muscle and was therefore called sTnTx. From both expression pattern and functional measurements, it appears that sTnT isoforms can be separated into two classes, high-molecular-weight (sTnT1, sTnT2) and low-molecular-weight (sTnTx, sTnT3) isoforms. By comparison to the apparent migration pattern of the four recombinant sTnT isoforms, the newly described low-molecular-weight sTnTx isoform appeared predominantly and typically expressed in fast skeletal muscles, whereas the higher-molecular-weight isoforms were more abundant in slow soleus muscle. The relative proportion of the sTnT isoforms in the soleus was not modified after exposure to hindlimb unloading (HU), known to induce a functional atrophy and a slow-to-fast isoform transition of several myofibrillar proteins. Functional data gathered from replacement of endogenous troponin complexes in skinned muscle fibers showed that the sTnT isoforms modified the Ca(2+) activation characteristics of single skeletal muscle fibers, with sTnT2 and sTnT1 conferring a similar increase in Ca(2+) affinity higher than that caused by low-molecular-weight isoforms sTnTx and sTnT3. Thus we show for the first time the presence of sTnT in fast muscle fibers, and our data show that the changes in neuromuscular activity on HU are insufficient to alter the sTnT expression pattern.

ADP-ribose stimulates the calcium release channel RyR1 in skeletal muscle of rat.

The Ca(2+) mobilizing metabolite cyclic ADP-ribose has been shown to release Ca(2+) from intracellular ryanodine sensitive stores in many cells. However, the activation of the ryanodine receptor of skeletal muscle by cADP-ribose (cADPr) and its precursor and metabolite (beta-NAD(+) and ADPr) remains to be discussed. We studied the effect of ADPr on the Ca(2+) release channel of skeletal muscle RyR1 after incorporation of microsomes isolated from fast muscles of rat in planar lipid bilayers. We observed an increase in the electrophysiological activity of the channel after addition of ADPr (10 microM) at micromolar Ca(2+) concentrations, characterized by a time-lag. The increase in P(o) is mainly due to an increase in the open frequency. The long time course observed for the development of the ADPr effect may indicate that this activation induces a change in the conformation of the RyR1 channel, which increases its sensitivity to calcium.

Expression and functional implications of troponin T isoforms in soleus muscle fibers of rat after unloading.

The expression pattern of troponin T (TnT) isoforms was studied in rat soleus muscle fibers in control and after hindlimb unloading (HU) conditions. To determine the functional consequence of TnT expression, the fibers were also examined for their calcium activation characteristics. With regard to TnT expression, four populations of fibers were distinguished in control muscle. Slow fibers expressing only slow isoforms of TnT (TnT1s, 2s, 3s ) were predominant (54%). Hybrid slow fibers (16%) differed from slow fibers by the additional expression of two TnTf isoforms. Hybrid fast fibers (22%) expressed slow and fast isoforms of TnT while fast fibers (8%) expressed only fast TnT isoforms. The expression of the other regulatory protein isoforms was checked for each population. The contractile experiments revealed steeper slopes of the tension/pCa relationship from hybrid slow fibers expressing fast TnT in a completely slow molecular environment. The expression of TnTs in hybrid fast fibers did not modulate the intrinsic co-operativity. After HU, the fast population was increased and reached 55%. The slow population decreased to 41% and a very small amount of hybrid slow fibers remained (4%). These data demonstrated the implication of TnT isoforms in the calcium activation properties and, more particularly, in the modulation of co-operativity within the myofibrillar lattice. Regulation of TnT expression appeared as a very fast and complete process compared to moderate changes of TnC and TnI.

Involvement of gap junctional communication and connexin expression in trophoblast differentiation of the human placenta.

Gap junctional intercellular communication (GJIC) permits coordinated cellular activities during development and differentiation processes, and its dysfunction or mutation of connexin genes have been implicated in pathologies. In the human placenta, two distinct differentiation pathways of cytotrophoblastic cell coexist leading to a double model: fusion phenotype (villous trophoblast) and proliferative/invasive phenotype (extravillous trophoblast). This review focuses on current knowledge on the connexin expression and the implication of GJIC in trophoblastic differentiation. Experimental evidence obtained in human placenta demonstrates the involvement of connexin 43-gap junctions in the trophoblastic fusion process and of a connexin switch during the spatially and temporally controlled proliferation/invasion process.

Expression and functional behavior of troponin C in soleus muscle fibers of rat after hindlimb unloading.

Troponin C (TnC) plays a key role in the regulation of muscle contraction, thereby modulating the Ca(2+)-activation characteristics of skinned muscle fibers. This study was performed to assess the effects of a 15-day hindlimb unloading (HU) period on TnC expression and its functional behavior in the slow postural muscles of the rat. We investigated the TnC isoform expression in whole soleus muscles and in single fibers. The latter were also checked for their Ca(2+) activation characteristics and sensitivity to bepridil, a Ca(2+) sensitizer molecule. This drug has been described as exerting a differential effect on slow and fast fibers, depending on the TnC isoform. With regard to TnC expression, three populations were found in control muscle fibers: slow, hybrid slow, and hybrid fast fibers, with the TnC fast being always coexpressed with TnC slow. In the whole muscle, TnC fast expression increased after HU because of the increase in the proportion of hybrid fast fibers. The HU hybrid fast fibers had properties similar to those of control hybrid fast fibers. The fibers that remained slow after HU exhibited similar bepridil and Sr(2+) properties as control slow fibers. Therefore, in these fibers, the changes could not be related to the TnC molecule.

The role of the Ca(2+) regulatory sites of skeletal troponin C in modulating muscle fibre reactivity to the Ca(2+) sensitizer bepridil.

1. The Ca(2+)-sensor protein troponin C (TnC) exerts a key role in the regulation of muscle contraction, and constitutes a target for Ca(2+) sensitizer compounds, such as bepridil, known to increase its apparent Ca(2+) affinity. Moreover, bepridil has been reported to exert a differential effect in slow and fast skeletal muscle fibres, which express the slow/cardiac and fast TnC isoform, respectively. 2. The role of the TnC isoform in establishing the differential effect of bepridil was assessed in slow soleus and fast tibialis rat skinned fibres, by extraction of endogenous TnC and consecutive reconstitution with either slow or fast recombinant TnC. A mutant (VG2), lacking the regulatory site II, was also used to distinguish the role of each regulatory site. 3. Fast tibialis fibres reconstituted with cardiac TnC exhibited a typical slow bepridil reactivity, while slow soleus fibres reincorporated with fast TnC displayed a typically fast reactivity to bepridil. These results indicated that the differential effect of bepridil in slow and fast fibres is related to the TnC isoform predominantly expressed in a fibre. 4. Experiments with the VG2 mutant demonstrated that BPD can achieve an increase in the Ca(2+) affinity in the absence of a functional site II. Thus, site I was necessary for the BPD effect to be independent of the Ca(2+) concentration. Moreover, the amplitude of the reinforcement in the Ca(2+) affinity, induced by the binding of bepridil to the TnC molecule, is dependent on the number of functional regulatory sites, the larger affinity reinforcement being detected when only one regulatory site (either site I or II) is functional.

Effects of unweighting and clenbuterol on myosin light and heavy chains in fast and slow muscles of rat.

To investigate the plasticity of slow and fast muscles undergoing slow-to-fast transition, rat soleus (SOL), gastrocnemius (GAS), and extensor digitorum longus (EDL) muscles were exposed for 14 days to 1) unweighting by hindlimb suspension (HU), or 2) treatment with the beta(2)-adrenergic agonist clenbuterol (CB), or 3) a combination of both (HU-CB). In general, HU elicited atrophy, CB induced hypertrophy, and HU-CB partially counteracted the HU-induced atrophy. Analyses of myosin heavy (MHC) and light chain (MLC) isoforms revealed HU- and CB-induced slow-to-fast transitions in SOL (increases of MHCIIa with small amounts of MHCIId and MHCIIb) and the upregulation of the slow MHCIa isoform. The HU- and CB-induced changes in GAS consisted of increases in MHCIId and MHCIIb ("fast-to-faster transitions"). Changes in the MLC composition of SOL and GAS consisted of slow-to-fast transitions and mainly encompassed an exchange of MLC1s with MLC1f. In addition, MLC3f was elevated whenever MHCIId and MHCIIb isoforms were increased. Because the EDL is predominantly composed of type IID and IIB fibers, HU, CB, and HU-CB had no significant effect on the MHC and MLC patterns.

Alterations in contractile properties and expression of myofibrillar proteins in wobbler mouse muscles.

The purpose of this study was to characterize the alterations in muscle contractile (tension-pCa relationship) and biochemical (myosin heavy and light chains, troponin C content) properties in a hereditary motoneuron disease. The study was performed on wobbler mouse mutants which presented a neuronal degeneration. The time course of the disease was followed at 5 and 7 weeks in sternocleidomastoid (SCM) and soleus muscles. The wobbler disease was found to induce a shift from fast to slow myosin heavy-chain isoform expression in SCM and soleus muscles. The analysis of the myosin light-chain (MLC) composition revealed, for the SCM muscles, the appearance of the slow isoforms at 5 weeks and an increase in the regulatory MLC2 content at 7 weeks. A significant increase in the slow troponin C isoform content was found in both types of wobbler muscles at 7 weeks. The wobbler soleus and SCM muscles presented an age- and fiber-type-related atrophy, characterized by a decline in absolute maximal tension and fiber diameter. A decrease in calcium sensitivity was observed at 7 weeks for the soleus fibers and at both 5 and 7 weeks for the SCM. The results indicated fast-to-slow changes in contractile and biochemical properties of the wobbler soleus and SCM muscles, which occurred during the motoneuron degeneration process previously described in the wobbler pathology.

Properties of ryanodine receptor in rat muscles submitted to unloaded conditions.

Unloading of skeletal muscles by hindlimb unweighting is known to induce muscle atrophy and a shift toward faster contractile properties associated with an increase in the expression of fast contractile proteins, particularly in slow soleus muscles. Contractile properties suggest that slow soleus muscles acquire SR properties close to those of a faster one. We studied the expression and properties of the sarcoplasmic reticulum calcium release (RyR) channels in soleus and gastrocnemius muscles of rats submitted to hindlimb unloading (HU). An increase in RyR1 and a slight decrease in RyR3 expression was detected in atrophied soleus muscles only after 4 weeks of HU. No variation appeared in fast muscles. [(3)H]Ryanodine binding experiments showed that HU neither increased the affinity of the receptors for [(3)H]ryanodine nor changed the caffeine sensitivity of [(3)H]ryanodine binding. Our results suggested that not only RyR1 but also RyR3 expression can be regulated by muscle activity and innervation in soleus muscle. The changes in the RyR expression in slow fibers suggested a transformation of the SR from a slow to a fast phenotype.

Single-channel properties of the sarcoplasmic reticulum calcium-release channel in slow- and fast-twitch muscles of Rhesus monkeys.

RyR1 is the main isoform of ryanodine receptor expressed in fast- and slow-twitch mammalian skeletal muscles although differences in Ca2+-release kinetics and properties have been reported. Single-channel measurements reveal that a large proportion (82%) of Ca2+-release channels measured in slow-twitch muscle preparations have properties similar to those of the Ca2+-release channels of fast-twitch preparations, i.e. the same conductance, an identical sensitivity to caffeine and a bell-shaped Ca2+ activation curve for pCa (-log10[Ca2+]) 7 to 3. A low proportion (18%) of Ca2+-release channels observed in preparations from slow-twitch muscles were characterized by a very high activity level. These channels were not inhibited at a millimolar concentration of Ca2+. Our data suggest that the different properties of Ca2+ release in slow- and fast-twitch muscles might not be related to intrinsic properties of the Ca2+-release channels of each type of muscle but rather to the co-expression of two isoforms of ryanodine receptor and the lower amount of Ca2+-release channels expressed in slow- than in fast-twitch muscles.

Uridine triphosphate-sensitive pathway of Ca2+ release from the sarcoplasmic reticulum of rat skeletal muscle fibers.

The pyrimidine nucleotide, uridine triphosphate (UTP), was tested with skinned skeletal muscle fibers in order to investigate the UTP-sensitive pathway of Ca2+ release from the sarcoplasmic reticulum. The presence of ryanodine (200 microM), ruthenium red (10 microM) or heparin (2.5 mg/ml) did not affect the tension elicited in the presence of UTP, demonstrating that the UTP-induced Ca2+ release involved neither ryanodine nor inositol triphosphate-sensitive channels. Drugs such as compound 48/80 or cyclopiazonic acid used to inhibit Ca2+-ATPase in its reverse function appeared to be, respectively, non-specific or without any inhibitory effect on the tension induced by UTP. Finally, the UTP-induced tension as well as the trifluoperazine-induced tension were abolished in the presence of spermidine (50 mM), supporting the hypothesis that the UTP-sensitive pathway of the SR Ca2+ release might occur through the uncoupled calcium ATPase.

Effect of bovine serum albumin on the calcium release channel of sarcoplasmic reticulum from rabbit skeletal muscle.

The effect of bovine serum albumin (BSA) on the activity of the calcium release channel of the sarcoplasmic reticulum from rabbit skeletal muscle was investigated using both tension recording from skinned fibres and electrophysiological recording of unitary channel currents from planar lipid membranes. BSA had no effect on the Ca2+ affinity of the contractile proteins, elicited no tension per se in Ca(2+)-loaded skinned fibres, but potentiated caffeine-induced tension. Maximum potentiation was observed with 0.05-0.5% BSA. BSA (0.1%) had no detectable effect on the basal activity of the Ca(2+)-release channel incorporated in lipid bilayer. However, channel stimulation elicited by either caffeine (2 mM) or ATP (60 microM) was further enhanced by BSA (0.1%), as indicated by significant increases in Po, the open probability of the channel. These results suggest that BSA can modulate the response of the skeletal muscle SR Ca(2+)-release channel to different activators such as caffeine and ATP.

Effect of SR33805 on barium current and asymmetric intramembrane charge movement in freshly dissociated mouse cerebellar Purkinje neurons.

SR33805 is a novel calcium channel blocker that binds selectively and with high affinity to the alpha 1 subunit of the L-type calcium channel. The binding site for SR33805 is distinct from other classical calcium channel blockers although they interact allosterically. The block by SR33805 of the neuronal L-type calcium current has been reported [Romey, G. and Lazdunski, M., J. Pharmacol. Exp. Ther., 271 (1994) 1348-1352.]. In Purkinje neurons, the L-type calcium current is nearly absent. Nevertheless, we have shown the presence of intramembrane charge movement related to the dihydropyridines (DHP) receptor in these neurons. We show here that SR33805 has no effect on barium currents recorded in Purkinje cells but is a very potent blocker of intramembrane charge movement. It reduces charge movement to 48% of control with an IC50 of 0.5 nM.

Contraceptive gossypol blocks cell-to-cell communication in human and rat cells.

Gossypol (a polycyclic lipophilic agent naturally present in cottonseed, known as a potent non-steroid antifertility agent and a non-specific enzyme inhibitor) irreversibly impaired the intercellular communication between homologous pairs of various cultured cells, from man or rat, involved (Sertoli or trophoblastic cells) or not involved (ventricular myocytes) in steroidogenesis, in a dose-dependent manner. In serum-free assays, a rapid junctional uncoupling occurred in non-cytotoxic conditions. At 5 microM (approximately twice the peak plasma concentration measured in human patients during chronic administration), gap junctional communication was interrupted within 4 to 10 min, without concomitant rise in the intracellular Ca2+ concentration. The latter importantly increased when gossypol treatment was prolonged (cytotoxic effect). The short term uncoupling effect of gossypol was prevented by serum proteins, but long-lasting treatments (48 h) with moderate concentrations (3 microM) elicited junctional uncoupling and impeded the in vitro differentiation of human trophoblasts.

Effect of antipeptide antibodies directed against three domains of connexin43 on the gap junctional permeability of cultured heart cells.

Cell-to-cell communication can be blocked by intracellular injections of antibodies raised against gap junction proteins, but the mechanism of channel obstruction is unknown. Binding to connexins could lead to a conformational change, interfere with regulatory domains or cause a steric hindrance. To address these questions, the effects on cell-to-cell communication of affinity purified polyclonal antibodies raised against peptides reproducing the intracellular sequences 5-17, 314-322 and 363-382 of rat connexin43 were investigated in cultured rat ventricular cells. The antibodies against sequence 363-382 were characterized by immunoblotting and immunocytochemistry. Characterization of antibodies 5-17 and 314-322 has been previously reported. In a first series of experiments, the effect on gap junctional communication was assessed by injecting a junction-permeant fluorescent dye into cells adjacent to one cell previously microinjected with antibodies. In a second series, junctional permeability was quantitatively determined on records of fluorescence recovery after the photobleaching of 6-carboxyfluorescein-loaded cells. Antibodies 5-17 marked a 43 kDa band on immunoblots, but did not immunolabel gap junctions and had no functional effect. Antibodies 314-322 recognized the 43 kDa protein and labeled the intercalated disks, but failed to interfere with junctional permeability. Antibodies to the nearby sequence 363-382, for which all immunospecific tests had been positive, caused a delayed diffusional uncoupling in 50% of the microinjected cells. It is suggested that the blocking of junctional communication by antibodies results from interference with a regulatory domain of the connexin.