Neonatal Muscle Research Articles

Developmental and Tissue-Specific Involvement of Peroxisome Proliferator-Activated Receptor-α in the Control of Mouse Uncoupling Protein-3 Gene Expression

Uncoupling protein-3 (UCP3) is a member of the mitochondrial carrier family expressed preferentially in skeletal muscle and heart. It appears to be involved in metabolic handling of fatty acids in a way that minimizes excessive production of reactive oxygen species. Fatty acids are powerful regulators of UCP3 gene transcription. We have found that the role of peroxisome proliferator-activated receptor-alpha (PPARalpha) on the control of UCP3 gene expression depends on the tissue and developmental stage. In adults, UCP3 mRNA expression is unaltered in skeletal muscle from PPARalpha-null mice both in basal conditions and under the stimulus of starvation. In contrast, UCP3 mRNA is down-regulated in adult heart both in fed and fasted PPARalpha-null mice. This occurs despite the increased levels of free fatty acids caused by fasting in PPARalpha-null mice. In neonates, PPARalpha-null mice show impaired UCP3 mRNA expression in skeletal muscle in response to milk intake, and this is not a result of reduced free fatty acid levels. The murine UCP3 promoter is activated by fatty acids through either PPARalpha or PPARdelta but not by PPARgamma or retinoid X receptor alone. PPARdelta-dependent activation could be a potential compensatory mechanism to ensure appropriate expression of UCP3 gene in adult skeletal muscle in the absence of PPARalpha. However, among transcripts from other PPARalpha and PPARdelta target genes, only those acutely induced by milk intake in wild-type neonates were altered in muscle or heart from PPARalpha-null neonates. Thus, PPARalpha-dependent regulation is required for appropriate gene regulation of UCP3 as part of the subset of fatty-acid-responsive genes in neonatal muscle and heart.

Endothelin-1 Inhibits Apoptosis of Pulmonary Arterial Smooth Muscle in the Neonatal Rat

Vascular wall remodeling in pulmonary hypertension is contributed to by an aberration in the normal balance between proliferation and apoptosis of smooth muscle. We observed that endothelin (ET)-1 is a critical mediator of vascular remodeling in neonatal rats chronically exposed to 60% O(2), but has no direct proliferative effects on cultured neonatal rat pulmonary artery smooth muscle cells (PASMCs). These findings led us to hypothesize that ET-1 may modulate remodeling by inhibiting apoptosis of smooth muscle. ET-1 (0.1 microM) was found to significantly attenuate both Paclitaxel- and serum deprivation-induced PASMC apoptosis, likely through stimulation of the ET(A) receptor (ET(A)R). ET-1 also prevented Paclitaxel-induced up-regulation of pro-apoptotic Bax and cleaved (activated) caspase-3. In rat pups exposed from birth to 60% O(2) for 7 d, arterial wall expression of Bax was decreased and expression of both ET(A)R and anti-apoptotic Bcl-xL were increased. Furthermore, increased numbers of TUNEL-positive cells were evident in the walls of pulmonary arteries from 60% O(2)-exposed animals treated with a combined ET receptor antagonist, SB217242, relative to air-exposed and vehicle-treated groups. Together, these findings suggest that ET-1 mediates remodeling of neonatal rat pulmonary arteries by inhibiting smooth muscle apoptosis.

Novel Geometries for Tissue-Engineered Tendonous Collagen Constructs

A promising approach to addressing the performance limitations of currently available mechanical and bioprosthetic heart valves lies in tissue engineering. Tissue-engineered valves should incorporate the complex microstructure of the native valves to mimic their unique mechanics. This would include a layered topology, mesh networks, and branched collagen fiber bundles. Our approach to heart valve tissue engineering is to develop the functional components of the aortic valve cusps separately in vitro and, once they are mature, integrate them into a composite valve structure. Here we report on our efforts to create more complex collagenous structures, suitable for heart valve tissue engineering. Collagen fiber bundles were fabricated using the principle of directed collagen gel contraction, using neonatal rat aortic smooth muscle cells and acid-soluble type I rat-tail tendon collagen. The collagen gels were cast into rectangular or branched wells with porous end holders that constrained the gels longitudinally but allowed contraction to occur transverse to the long axis. Pairs of such constructs were placed in direct contact with each other and cultured further to determine whether they integrated to form continuous tissue. After 6-8 weeks of culture, highly compacted and aligned collagen fiber bundles formed. Mechanical testing revealed that linear constructs (2 free ends) with an 8:1 aspect ratio were significantly stronger than similar constructs with an aspect ratio of 2:1 (mean +/- SD, 298 +/- 90 kPa vs. 152 +/- 49 kPa; p < .001). Branching reduced mechanical strength considerably. Constructs fabricated with 4 free ends were significantly weaker than constructs with 3 ends (31 +/- 32 kPa vs. 116 +/- 66 kPa; p < .003). Histologic images demonstrated the integration of the crossed collagen bundles, with a bonding strength of 2.1 +/- 1.1 g (0.02 N). We found that the geometry of the molds into which the collagen constructs are cast can greatly affect their mechanical strength: multibranched constructs were the weakest, and long, linear constructs were the strongest. We also found that integration of collagen constructs occurs in vitro and that the fabrication of a composite structure in vitro is probably feasible.

C-Terminal-Truncated Microdystrophin Recruits Dystrobrevin and Syntrophin to the Dystrophin-Associated Glycoprotein Complex and Reduces Muscular Dystrophy in Symptomatic Utrophin/Dystrophin Double-Knockout Mice

C-terminal-truncated (ΔC) microdystrophin is being developed for Duchenne muscular dystrophy gene therapy. Encouraging results have been achieved in the mdx mouse model. Unfortunately, mdx mice do not display the same phenotype as human patients. Evaluating ΔC microdystrophin in a symptomatic model will be of significant relevance to human trials. Utrophin/dystrophin double-knockout (u-dko) mice were developed to model severe dystrophic changes in human patients. In this study we evaluated the therapeutic effect of the ΔR4-R23/ΔC microdystrophin gene (ΔR4/ΔC) after serotype-6 adeno-associated virus-mediated gene transfer in neonatal u-dko muscle. At 2 months after gene transfer, the percentage of centrally nucleated myofiber was reduced from 89.2 to 3.4% and muscle weight was normalized. Furthermore, we have demonstrated for the first time that ΔC microdystrophin can eliminate interstitial fibrosis and macrophage infiltration and restore dystrobrevin and syntrophin to the dystrophin-associated glycoprotein complex. Interestingly neuronal nitric oxide synthase was not restored. The most impressive results were achieved in muscle force measurement. Neonatal gene therapy increased twitch- and tetanic-specific force. It also brought the response to eccentric contraction-induced injury to the normal range. In summary, our results suggest that the ΔR4/ΔC microgene holds great promise in preventing muscular dystrophy.

Induction of Neuregulin Signaling in Mouse Schwann Cells In Vivo Mimics Responses to Denervation

Neuregulins play crucial roles in early development of Schwann cells (SCs), but their roles in the activities of SCs during denervation and reinnervation of muscle are less clear. In the present study, the Tet-On system has been used in transgenic mice to enable inducible expression of a mutant, constitutively active neuregulin receptor (ErbB2) in SCs. This induction simulates neuregulin signaling to these cells. Reporter transgenes were used to show a tightly regulated, SC-selective expression in muscle. Induction leads to a number of changes in SCs at neuromuscular junctions that mimic the response to muscle denervation/reinnervation. These include process extension, soma migration, and proliferation. SCs also come to express nestin, a protein characteristic of their reaction to muscle denervation. This activation of SCs results in the sprouting of nerve terminals, and these sprouts follow the extensions of the SCs. However, these sprouts and their associated SCs disappear after the removal of the inducer. Last, induction of the active receptor is sufficient to rescue SCs in neonatal muscle from denervation-induced apoptosis. These findings show that the responses of SCs in muscle to denervation can be explained by induction of an autocrine/paracrine neuregulin signaling cascade suggested by previous molecular studies.

Hypertrophic response of Duchenne and limb-girdle muscular dystrophies is associated with activation of Akt pathway

Dystrophic muscle undergoes repeated cycles of degeneration/regeneration, characterized by the presence of hypertrophic fibers. In order to elucidate the signaling pathways that govern these events, we investigated Akt activation in normal and dystrophic muscle. Akt is activated in neonatal muscle and in actively dividing myoblasts, supporting a developmental role for Akt signaling. Akt activation was detected at very early, prenecrotic stages of disease pathogenesis, and maximal activation was observed during peak stages of muscle hypertrophy. Duchenne muscular dystrophy patients exhibit a similar pattern of Akt activation. Mice with sarcoglycan-deficient muscular dystrophy possess more severe muscle pathology and display elevated Akt signaling. However, the highest levels of Akt activation were found in dystrophin–utrophin-deficient muscle with very advanced dystrophy. We propose that Akt may serve as an early biomarker of disease and that Akt activation mediates hypertrophy in muscular dystrophy. Current investigations are focused on introducing constitutively active and dominant-negative Akt into prenecrotic mdx mice to determine how early modification of Akt activity influences disease pathogenesis.

Creation of Engineered Cardiac Tissue In Vitro From Mouse Embryonic Stem Cells

Embryonic stem (ES) cells can terminally differentiate into all types of somatic cells and are considered a promising source of seed cells for tissue engineering. However, despite recent progress in in vitro differentiation and in vivo transplantation methodologies of ES cells, to date, no one has succeeded in using ES cells in tissue engineering for generation of somatic tissues in vitro for potential transplantation therapy. ES-D3 cells were cultured in a slow-turning lateral vessel for mass production of embryoid bodies. The embryoid bodies were then induced to differentiate into cardiomyocytes in a medium supplemented with 1% ascorbic acid. The ES cell-derived cardiomyocytes were then enriched by Percoll gradient centrifugation. The enriched cardiomyocytes were mixed with liquid type I collagen supplemented with Matrigel to construct engineered cardiac tissue (ECT). After in vitro stretching for 7 days, the ECT can beat synchronously and respond to physical and pharmaceutical stimulation. Histological, immunohistochemical, and transmission electron microscopic studies further indicate that the ECTs both structurally and functionally resemble neonatal native cardiac muscle. Markers related to undifferentiated ES cell contamination were not found in reverse transcriptase-polymerase chain reaction analysis of the Percoll-enriched cardiomyocytes. No teratoma formation was observed in the ECTs implanted subcutaneously in nude mice for 4 weeks. ES cells can be used as a source of seed cells for cardiac tissue engineering. Additional work remains to demonstrate engraftment of the engineered heart tissue in the case of cardiac defects and its functional integrity within the host's remaining healthy cardiac tissue.

Sarcomeric Alpha‐actinin is a Myotendinous Junction Component in Neonatal Mice

Myotendinous junctions (MTJs) are cell adhesion sites where new sarcomeres are added in series during longitudinal muscle growth. Currently, little is known about the developmental dynamics of this specialized region of the muscle cell. This study compared the ultrastructure and composition of MTJs from rapidly-growing neonatal mice, and adult mice whose muscles have reached a steady-state length. At neonatal MTJs membrane exhibits greatly reduced folding, myofibrils are immature and interspersed with unorganized cytoplasm, and the tendon is densely populated with mononucleated cells, most likely fibroblasts, many of which are in close association with the muscle cell. Immunolocalization of the nebulin-related protein N-RAP, which is localized exclusively to the terminal cytoskeletal filament bundles at MTJs in adult skeletal muscle, is also restricted to these sites in neonatal muscle. Vinculin and dystrophin were co-localized subjacent to the membrane, also at both adult and neonatal MTJs. Interestingly, sarcomeric α-actinin was localized to MTJ adhesion plaques in neonates, but was not detected at the adult MTJ. The variable presence of sarcomeric α-actinin at MTJs suggests that muscle fibers differentially regulate actin-membrane interactions during postnatal growth, either in response to developmental stage-specific differences in mechanical stress, or to accommodate serial sarcomere addition in neonates. Supported by a grant from the American Heart Association, and by the Dean of the UMKC School of Biological Sciences.

Myosin phosphatase isoform switching in vascular smooth muscle development

We are using the myosin phosphatase targeting subunit (MYPT1) as a model gene to study smooth muscle phenotypic diversity. Myosin phosphatase (MP) is the primary effector of smooth muscle relaxation, and MYPT1 is a key target of signals that regulate smooth muscle tone. In a model of portal hypertension we previously showed dynamic changes in the expression of MYPT1 isoforms in the portal vein and upstream mesenteric artery. We hypothesized that this represents a reversion to the fetal phenotype characteristic of muscle hypertrophy. Here we studied MP during vascular smooth muscle phenotypic specification. Between postnatal days 6 and 12 the expression of MYPT1 increased approximately twofold in portal vein with a similar increase in MP activity. MYPT1 switched from C-terminal leucine zipper (LZ) positive to LZ negative splice variant isoforms. This was concordant with a switch from sensitive (10 −7 M) to resistant to cGMP-mediated vascular relaxation. This is consistent with the model in which the MYPT1 C-terminal LZ is required for cGMP-dependent activation of MP. Concordant changes in the expression of other contractile proteins were consistent with a switch from a slow-tonic to a fast-phasic contractile phenotype. In contrast aortic smooth muscle throughout development expressed the MYPT1 LZ positive isoform and relaxed to cGMP. We propose that MP isoform switching during neonatal vascular smooth muscle phenotypic specification may determine changing vascular responses to NO/cGMP signaling in the transition from the fetal to the adult circulation.

Quantification by real-time PCR of developmental and adult myosin mRNA in rat muscles

A real-time RT-PCR assay using newly designed primers was developed to analyze developmental and adult MHC mRNA expression both in skeletal muscles and single fibers. Only 4 ng of total RNA was necessary for the analysis of the relative mRNA expression of MHC genes. Different validation steps were realized concerning both specificity and sensitivity of each primer set, and linearity and efficiency of each real-time PCR amplification. Then, quantification of MHC mRNA in neonatal and adult muscles as well as in single fibers was done by the Δ C T method, with CycA gene as the reference gene. Due to a higher sensitivity than that of a competitive PCR method, we demonstrated that this assay is suitable to study very low level of MHC mRNA expression as developmental MHC in adult muscle and to quantify mRNA from very small samples.

Mechanism of Endothelin-1 Activation of Map Kinases in Neonatal Pulmonary Vascular Smooth Muscle

Mitogen-activated protein kinases (MAPKs) belong to the group of serine-threonine kinases that are rapidly activated in response to growth factor stimulation. In adult mammalian cells, the MAPK family includes extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2 or p44(mapk) and p42(mapk)), which translocate to the nucleus and integrate signals from second messengers leading to cellular proliferation or differentiation, but the specific role of MAPKs in neonatal pulmonary vascular smooth muscle is not well understood. Expression of p44(mapk) and p42(mapk) in primary cultured pulmonary vascular smooth muscle cells from neonatal (1-2 day old) rats was identified by Western immunoblot analysis and treatment with 10 nM endothelin-1 (ET-1), a potent vasoconstrictor with vascular mitogenic properties, induced cell proliferation, and phosphorylation of both p44(mapk) and p42(mapk). The protein kinase C (PKC) isozyme inhibitor (alpha, beta, gamma, delta, zeta) Go 6983, the ET(A) receptor antagonist BQ 123, and the MAPK kinase inhibitor PD98059 blocked the cell proliferation response to ET-1. Also, BQ 123, Go 6983, and PKC inhibitor 20-28 (Myr-N-FARKGAL-RQ-NH2-PKCalpha antagonist) inhibited ET-1-induced phosphorylation of both p44(mapk) and p42(mapk). In contrast, the reactive oxygen species (ROS) inhibitor diphenylene iodonium (DPI), the PKCdelta inhibitor rottlerin, and the ET(B) receptor antagonist BQ 788 did not block ET-1-induced phosphorylation of MAPKs. Collectively, these data demonstrate the expression and phosphorylation of MAPKs by ET-1 and suggests that MAPK activation and cell proliferation by ET-1 occurs via ET(A) receptor stimulation and specific PKC isozyme activation in rat neonatal pulmonary vascular smooth muscle.

Effect of Nitric Oxide on Mitogen-Activated Protein Kinases in Neonatal Pulmonary Vascular Smooth Muscle

Mitogen-activated protein kinases (MAPKs) belong to the group of serine/threonine kinases that are rapidly activated in response to growth factor stimulation. In adult mammalian cells, the MAPK family includes extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2 or p44mapk and p42mapk), which translocate to the nucleus and integrate signals from second messengers leading to cellular proliferation or differentiation. However, the specific role of MAPKs in neonatal pulmonary vascular smooth muscle is not well understood. Expression of p44mapk and p42mapk in primary cultured pulmonary vascular smooth muscle cells from neonatal (1-2 day old) rats was identified by Western immunoblot analysis. Treatment with 10 nM endothelin-1 (ET-1), a potent vasoconstrictor with vascular mitogenic properties, induced phosphorylation of both p44mapk and p42mapk, but treatment with the exogenous nitric oxide (NO) donor sodium nitroprusside inhibited both p44mapk and p42mapk phosphorylation by ET-1. The specific cGMP-dependent protein kinase (PKG) inhibitor KT5823, the nonspecific nitric oxide synthase (NOS) inhibitor L-NAME, and the specific NOS 1 blocker NPLA all significantly enhanced both p44mapk and p42mapk phosphorylation by ET-1. Collectively, these data demonstrate the expression and phosphorylation of specific MAPKs in rat neonatal pulmonary vascular smooth muscle and suggests that the NO signaling pathway modulates MAPK activation by ET-1.

Selective expression of the type 3 isoform of ryanodine receptor Ca 2+ release channel (RyR3) in a subset of slow fibers in diaphragm and cephalic muscles of adult rabbits

The expression pattern of the RyR3 isoform of Ca 2+ release channels was analysed by Western blot in neonatal and adult rabbit skeletal muscles. The results obtained show that the expression of the RyR3 isoform is developmentally regulated. In fact, RyR3 expression was detected in all muscles analysed at 2 and 15 days after birth while, in adult animals, it was restricted to a subset of muscles that includes diaphragm, masseter, pterygoideus, digastricus, and tongue. Interestingly, all of these muscles share a common embryonic origin being derived from the somitomeres or from the cephalic region of the embryo. Immunofluorescence analysis of rabbit skeletal muscle cross-sections showed that RyR3 staining was detected in all fibers of neonatal muscles. In contrast, in those adult muscles expressing RyR3 only a fraction of fibers was labelled. Staining of these muscles with antibodies against fast and slow myosins revealed a close correlation between expression of RyR3 and fibers expressing slow myosin isoform.

Multiple alternatively spliced transcripts of the receptor tyrosine kinase MuSK are expressed in muscle

We have cloned and characterized five distinct mouse cDNAs encoding isoforms of MuSK, a receptor tyrosine kinase required for the development of the neuromuscular synapse. Comparison of the cDNA sequences with each other and with the mouse genomic sequence revealed that the cDNAs differ by the presence or absence of three alternatively spliced exons encoding an additional 10, 15 and 8 amino acids respectively. The location and sequences of these exons are conserved in MuSK cDNAs from different species. Examination of mouse genomic sequences revealed that the 15 aa exon sequence is present in a 52 bp exon with a non-canonical 3′ splice acceptor site at its 5′ end and an internal 3′ splice acceptor site consensus 45 bp downstream. Transcripts containing each of the alternatively spliced exons were detected in neonatal and adult mouse muscle, as well as in C2 myotubes. The presence of transcripts encoding MuSK isoforms with distinct extracellular domains in developing mouse muscle suggests that alternative splicing could potentially introduce additional complexity to the activity of MuSK in muscle.

Expression of Slow Skeletal Troponin I in Adult Mouse Heart Helps to Maintain the Left Ventricular Systolic Function During Respiratory Hypercapnia

Compared with the adult, neonatal heart muscle is less sensitive to deactivation by acidic pH. We hypothesized that expression of slow skeletal troponin I (ssTnI), the embryonic isoform, in adult heart would help maintain left ventricular (LV) systolic function during respiratory hypercapnia. We assessed LV function by transthoracic 2D-targeted M-mode and pulsed Doppler echocardiography in transgenic (TG) mice in which cardiac TnI was replaced with ssTnI and in nontransgenic (NTG) littermates. Anesthetized mice were ventilated with either 100% oxygen or 35% CO2 balanced with oxygen. Arterial blood pH with 35% CO2 decreased to the same levels in both groups of animals. In the absence of propranolol, the LV fractional shortening was higher in TG compared with NTG mice throughout most of the experimental protocol. LV diastolic function was impaired in TG compared with NTG mice both at 100% oxygen and 35% CO2 because E-to-A wave ratio of mitral flow was significantly lower, and E-wave deceleration time and LV isovolumic relaxation time were longer in TG compared with NTG mice. When compensatory mechanisms that occur through stimulation of beta-adrenergic receptors during hypercapnia were blocked by continuous perfusion with propranolol, we found that NTG mice died within 3 to 4 minutes after switching to 35% CO2, whereas TG mice survived. Our experiments demonstrate the first evidence that specific replacement of cardiac TnI with ssTnI has a protective effect on the LV systolic function during hypercapnic acidosis in situ.

A method to establish co-cultures of myotubes and preadipocytes from collagenase digested neonatal pig semitendinosus muscles1

The relationships between adipocyte and muscle cell development within muscle are important in the study of factors or agents that may improve meat quality. Neonatal porcine muscle has the potential to yield both cell types for cell culture because it contains developing adipocytes and a high number of muscle satellite cells. Therefore, we modified a conventional collagenase-based procedure to digest neonatal porcine muscle and subsequently cultured the resultant muscle stromal-vascular (SV) cells on several substrata in basal and dexamethasone (DEX)-containing media. Developing myotubes and preadipocytes were present in muscle SV cell cultures on laminin substrata following seeding and plating with fetal bovine serum (FBS) with or without DEX. Myotube number was much higher (P < 0.05) on laminin substrata compared with all other substrata, whereas preadipocyte number in muscle SV cell cultures was independent of substrata, as we have shown previously. This approach can be used to establish co-cultures of differentiating adipocytes and myotubes from collagenase-digested neonatal pig muscle. Because the comparison is within the same culture dish, this method allows for a direct comparison of the responses of adipogenic and myogenic cells to growth and differentiation factors. For example, DEX did not alter myogenesis (i.e., 11 +/- 3 vs. 11 +/- 4 myotubes per unit area for control and DEX-treated cultures, respectively), but it has been shown to markedly increase preadipocyte number in muscle SV cell cultures.

Evaluation of the cardioprotective potential of Buckberg vs. Bretschneider cardioplegia in neonatal pig left-ventricle cardiac muscle tissue

Evaluation of the cardioprotective potential of Buckberg vs. Bretschneider cardioplegia in neonatal pig left-ventricle cardiac muscle tissue

Physiological rise in plasma leucine stimulates muscle protein synthesis in neonatal pigs by enhancing translation initiation factor activation

Protein synthesis in skeletal muscle of adult rats increases in response to oral gavage of supraphysiological doses of leucine. However, the effect on protein synthesis of a physiological rise in plasma leucine has not been investigated in neonates, an anabolic population highly sensitive to amino acids and insulin. Therefore, in the current study, fasted pigs were infused intra-arterially with leucine (0, 200, or 400 micromol.kg(-1).h(-1)), and protein synthesis was measured after 60 or 120 min. Protein synthesis was increased in muscle, but not in liver, at 60 min. At 120 min, however, protein synthesis returned to baseline levels in muscle but was reduced below baseline values in liver. The increase in protein synthesis in muscle was associated with increased plasma leucine of 1.5- to 3-fold and no change in plasma insulin. Leucine infusion for 120 min reduced plasma essential amino acid levels. Phosphorylation of eukaryotic initiation factor (eIF)-4E-binding protein-1 (4E-BP1), ribosomal protein (rp) S6 kinase, and rpS6 was increased, and the amount of eIF4E associated with its repressor 4E-BP1 was reduced after 60 and 120 min of leucine infusion. No change in these biomarkers of mRNA translation was observed in liver. Thus a physiological increase in plasma leucine stimulates protein synthesis in skeletal muscle of neonatal pigs in association with increased eIF4E availability for eIF4F assembly. This response appears to be insulin independent, substrate dependent, and tissue specific. The results suggest that the branched-chain amino acid leucine can act as a nutrient signal to stimulate protein synthesis in skeletal muscle of neonates.

Effects of elastase on the mechanical and failure properties of engineered elastin-rich matrices

Pulmonary emphysema and vessel wall aneurysms are diseases characterized by elastolytic damage to elastin fibers that leads to mechanical failure. To model this, neonatal rat aortic smooth muscle cells were cultured, accumulating an extracellular matrix rich in elastin, and mechanical measurements were made before and during enzymatic digestion of elastin. Specifically, the cells in the cultures were killed with sodium azide, the cultures were lifted from the flask, cut into small strips, and fixed to a computer-controlled lever arm and a force transducer. The strips were subjected to a broadband displacement signal to study the dynamic mechanical properties of the samples. Also, quasi-static stress-strain curves were measured. The dynamic data were fit to a linear viscoelastic model to estimate the tissues' loss (G) and storage (H) modulus coefficients, which were evaluated before and during 30 min of elastase treatment, at which point a failure test was performed. G and H decreased significantly to 30% of their baseline values after 30 min. The failure stress of control samples was approximately 15 times higher than that of the digested samples. Understanding the structure-function relationship of elastin networks and the effects of elastolytic injury on their mechanical properties can lead to the elucidation of the mechanism of elastin fiber failure and evaluation of possible treatments to enhance repair in diseases involving elastolytic injury.

Increased relaxation of immature airways to β2-adrenoceptor agonists is related to attenuated expression of postjunctional smooth muscle muscarinic M2 receptors

Spontaneous or agonist-induced contraction of airway smooth muscle can be observed very early in fetal life, thus explaining the possible occurrence of bronchospasm in very low birth weight infants within the first days of life. In an attempt to better manage such bronchospasms, the aim of the present study was to investigate the age-specific modifications in airway smooth muscle relaxation to beta2-agonists and muscarinic antagonists using a combination of functional and molecular techniques. In the rat, isometric relaxation to the beta2-agonist salbutamol was examined in tracheae; we also examined muscarinic receptor expression (M2R and M3R mRNA levels) in airway smooth muscle by immunochemistry, Western blotting, and real-time PCR. Compared with adults, salbutamol-induced relaxation was twofold greater in immature rat isolated tracheae preconstricted by carbachol. This effect was associated with a lower expression of M2R in the smooth muscle of immature animals (sixfold and almost twofold as assessed by immunochemistry and Western blotting, respectively). Real-time PCR data indicate that changes in M2R expression according to age occurred at a posttranscriptional level. In adult airways, there was a significantly greater functional efficacy of M2R blockade by methoctramine compared with that shown in immature rats. Because of the limited availability of human neonate lung tissue, only the molecular part of the study was performed, and we observed a qualitatively similar effect, i.e., a lower M2R expression in the neonatal airway smooth muscle, although this was quantitatively smaller. We conclude that beta2-agonist-induced relaxation is enhanced in immature compared with adult airways as a result of greater postjunctional M2R expression in adult airway smooth muscle. This finding may be of importance in the clinical management of bronchoconstriction in neonates.