Bone biomechanical properties in LRP5 mutant mice.

The mutation responsible for the high bone mass (HBM) phenotype has been postulated to act through the adaptive response of bone to mechanical load resulting in denser and stronger skeletons in humans and animals. The bone phenotype of members of a HBM family is characterized by normally shaped bones that are exceptionally dense, particularly at load bearing sites [Cancer Res. 59 (1999) 1572]. The high bone mass (HBM) mutation was identified as a glycine to valine substitution at amino acid residue 171 in the gene coding for low-density lipoprotein receptor-related protein 5 (LRP5) [Bone Miner. Res. 16(4) (2001) 758]. Thus, efforts have focused on the examination of the role of LRP5 and the G171V mutation in bone mechanotransduction responses [J. Bone Miner. Res 18 (2002) 960]. Transgenic mice expressing the human G171V mutation have been shown to have skeletal phenotypes remarkably similar to those seen in affected individuals. In this study, we have identified differences in biomechanical (structural and apparent material) properties, bone mass/ash, and bone stiffness of cortical and cancellous bone driven by the G171V mutation in LRP5. As in humans, the LRP5 G171V plays an important role in regulating bone structural phenotypes in mice. These bone phenotypes include greater structural and apparent material properties in HBM HET as compared to non-transgenic littermates (NTG) mice. Body size and weight in HBM HET were similar to that in NTG control mice. However, the LRP5 G171V mutation in HET mice results in a skeleton that has greater structural (femoral shaft, femoral neck, tibiae, vertebral body) and apparent material (vertebral body) strength, percent bone ash weight (ulnae), and tibial stiffness. Despite similar body weight to NTG mice, the denser and stiffer bones in G171V mice may represent greater bone formation sensitivity to normal mechanical stimuli resulting in an overadaptation of skeleton to weight-related forces.

Postnatal expression in hyaline cartilage of constitutively active human collagenase-3 (MMP-13) induces osteoarthritis in mice.

It has been suggested that increased collagenase-3 (MMP-13) activity plays a pivotal role in the pathogenesis of osteoarthritis (OA). We have used tetracycline-regulated transcription in conjunction with a cartilage-specific promoter to target a constitutively active human MMP-13 to the hyaline cartilages and joints of transgenic mice. Postnatal expression of this transgene resulted in pathological changes in articular cartilage of the mouse joints similar to those observed in human OA. These included characteristic erosion of the articular cartilage associated with loss of proteoglycan and excessive cleavage of type II collagen by collagenase, as well as synovial hyperplasia. These results demonstrate that excessive MMP-13 activity can result in articular cartilage degradation and joint pathology of the kind observed in OA, suggesting that excessive activity of this proteinase can lead to this disease.

The 2.3 kb smooth muscle myosin heavy chain promoter directs gene expression into the vascular system of transgenic mice and rabbits.

BACKGROUND: Smooth muscle cells (SMC) are a preferential target for gene therapeutic approaches in atherosclerosis and restenosis. However, the undesirable expression of putative therapeutic genes in tissues other than the vascular wall is a considerable safety limitation for clinical trials, thus requiring the identification of a smooth-muscle-specific promoter sequence. Since the 2.3 kb rabbit Smooth Muscle Myosin Heavy Chain (SMHC) promoter was shown to be transcriptionally active in primary vascular but not visceral or other non-SMC in vitro, this fragment was chosen for in vivo analysis. METHODS AND RESULTS: Transgenic mice and rabbits were established expressing a luciferase reporter gene under control of the 2.3 kb rabbit SMHC promoter. In contrast to the endogenous expression pattern of the SMHC gene both species revealed light emission predominantly in the arterial system including coronary arteries. Low activities were measured in large veins and the gastrointestinal system. In situ hybridization of murine embryos using a luciferase riboprobe confirmed reporter gene expression in large arteries with no detectable mRNA in the viscera. Unlike adult animals, ectopic luciferase activities were found in ventricular myocardium during murine development ceasing 1 week post partum. CONCLUSIONS: In two animal species, the 2.3 kb SMHC promoter appeared to be effective in discriminating between the pathways regulating vascular and visceral smooth muscle gene expression. The vascular-specific expression profile of the 2.3 kb SMHC promoter suggests that the 2.3 kb SMHC promoter contains the regulatory elements necessary for selective gene targeting into vascular SMC of large arteries including coronary arteries in vivo.

Inhibition of cardiac delayed rectifier K+ current by overexpression of the long-QT syndrome HERG G628S mutation in transgenic mice.

Mutations in the HERG gene are linked to the LQT2 form of the inherited long-QT syndrome. Transgenic mice were generated expressing high myocardial levels of a particularly severe form of LQT2-associated HERG mutation (G628S). Hearts from G628S mice appeared normal except for a modest enlargement seen only in females. Ventricular myocytes isolated from adult wild-type hearts consistently exhibited an inwardly rectifying E-4031-sensitive K+ current resembling the rapidly activating cardiac delayed rectifier K+ current (Ikr) in its time and voltage dependence; this current was not found in cells isolated from G628S mice. Action potential duration was significantly prolonged in single myocytes from G628S ventricle (cycle length=1 second, 26 degrees C) but not in recordings from intact ventricular strips studied at more physiological rates and temperature (200 to 400 bpm, 37 degrees C). ECG intervals, including QT duration, were unchanged, although minor aberrancies were noted in 20% (16/80) of the G628S mice studied, primarily involving the QRS complex and, more rarely, T-wave morphology. The aberrations were more commonly observed in females than males but could not be correlated with sex-based differences in action potential duration. These results establish the presence of IKr in the adult mouse ventricle and demonstrate the ability of the G628S mutation to exert a dominant negative effect on endogenous IKr in vivo, leading to the expected LQT2 phenotype of prolonged repolarization at the single cell level but not QT prolongation in the intact animal. The model may be useful in dissecting repolarization currents in the mouse heart and as a means of examining the mechanism(s) by which the G628S mutation exerts its dominant negative effect on native cardiac cells in vivo.

A novel smooth muscle-specific enhancer regulates transcription of the smooth muscle myosin heavy chain gene in vascular smooth muscle cells.

Transient DNA transfection analysis of 5' end deletion mutants of the rabbit smooth muscle myosin heavy chain (SMHC) gene promoter was performed in primary cultures of rabbit vascular smooth muscle cells (VSMC). A positive element located at position -1,332 upstream of the transcription start site consistently gave the highest relative chloramphenicol acetyltransferase (CAT) activity (6.3 +/- 1.5-fold over the minimal SMHC promoter), suggesting that inclusion of the extra 107-base pair (bp) DNA fragment between -1,332 and -1,225 could significantly enhance CAT activity in VSMC. Transfection of mutants into several muscle and nonmuscle cell lines did not show any significant CAT activity above control, showing that factors unique to smooth muscle cells were required for SMHC expression. Gel shift analysis indicated that multiple factors interacted with the 107-bp element, two of which appeared to show smooth muscle specificity. Tests of enhancer function in transfected VSMC indicated that the 107-bp fragment behaved as a classical enhancer, i.e. independently of position and orientation. These results indicate that a novel DNA element may regulate the tissue-restricted expression of the SMHC gene and provides the first example of a role for a smooth muscle-specific enhancer in VSMC.

Smooth muscle myosin regulation by serum and cell density in cultured rat lung connective tissue cells.

RNA and protein analyses were used to detect expression of SM1 and SM2 smooth muscle myosin heavy chain (MHC) in cultured adult rat lung connective tissue cells (RL-90). Smooth muscle MHC mRNA expression in confluent cells grown in 10% serum was approximately 50% of the level in adult stomach. Similar results were obtained in cells cultured at low density (25% confluency) in 1% serum. However, in low-density cultures transferred to 10% serum for 24 h, the level of MHC mRNA decreased to approximately 20% of that in adult stomach. Smooth muscle alpha-actin showed a pattern of expression similar to that for smooth muscle MHC. Expression of nonmuscle MHC-A mRNA was higher in all culture conditions compared to stomach. MHC-A mRNA expression was less in low-density cultures in low serum and increased when low-density cultures were transferred to 10% serum for 24 h. MHC-B mRNA expression was less in low- vs. high-density cultures. In contrast to MHC-A, however, MHC-B mRNA expression in low-density cultures was higher in low serum. Immunofluorescence and immunoblotting with SM1-specific antibody demonstrated the presence of the SM1 protein isoform as well as reactivity to a protein band migrating slightly faster than SM2. These results demonstrate that cultured rat lung connective tissue cells express smooth muscle MHC and that expression is modulated by culture conditions.

Tissue-specific and developmentally regulated alternative splicing of a visceral isoform of smooth muscle myosin heavy chain.

Previous work demonstrated that the rabbit smooth muscle myosin heavy chain gene showed sequence divergence at the 25kDa/50kDa junction of the S1 subfragment when compared to chicken gizzard and chicken epithelial nonmuscle myosin. RNase protection analysis with a probe spanning this region detected two partially protected fragments which were not present in RNA from vascular tissue and only found in RNA from visceral tissue. The polymerase chain reaction was used to amplify a 162bp product from primers spanning the putative region of divergence and DNA sequence analysis revealed a seven amino acid insertion not previously detected in other characterised cDNA clones. RNase protection analysis using the PCR product as probe showed that the inserted sequence was expressed exclusively in RNA from visceral tissue. Similar RNA analysis showed that the visceral isoform was not expressed in 20 day fetal rabbit smooth muscle tissues. These results indicated that the new visceral isoform was expressed in a tissue-specific and developmentally regulated manner. Genomic DNA sequencing and mapping of the exon-intron boundaries showed that the visceral isoform was the product of cassette-type alternative splicing. The inclusion of a visceral-specific sequence near the Mg-ATPase domain and at the 25kDa/50kDa junction suggests that the visceral isoform may be important for myosin function in smooth muscle cells.

Differential expression of SM1 and SM2 myosin isoforms in cultured vascular smooth muscle.

Ribonuclease protection assays were used to measure expression of smooth muscle (SM) specific myosin heavy chain (MHC) isoforms SM1 (204 kDa) and SM2 (200 kDa) and also nonmuscle MHC-A in cultured smooth muscle cells isolated from rat aorta. In cells grown in 10% serum for 3-5 days until subconfluent, SM1 MHC mRNA decreased by 30% and SM2 MHC mRNA decreased by 80%. In cells grown in confluency for 7-11 days, SM1 MHC mRNA decreased by 45% and SM2 MHC mRNA decreased by 80%. Similar reductions were found in passaged cells. Serum withdrawal for 1-2 days from confluent cultures had little or no effect on SM1 or SM2 MHC mRNA levels. In contrast, nonmuscle MHC-A mRNA increased 10-fold in subconfluent cultures but increased only threefold higher than controls in quiescent cells. Myosin protein analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting indicated that SM1 MHC protein was detectable at a reduced level in confluent cultured cells, whereas SM2 MHC protein was absent in confluent cells. The decrease in SM2 was much greater than SM1, indicating differential regulation. An apparently new isoform of SM1 MHC migrating with a mobility similar to SM2 type MHC was detected by immunoblot analysis in cultured cells.

Characterization of a mammalian smooth muscle myosin heavy-chain gene: complete nucleotide and protein coding sequence and analysis of the 5' end of the gene.

The purpose of this study was to characterize the complete cDNA sequence encoding the rabbit smooth muscle myosin heavy chain (MHC) and determine the exon/intron organization at the 5' end of the corresponding gene. The full-length cDNA sequence of 6644 base pairs encoding a protein of 1972 amino acids was generated from two cDNA clones: PBRUC1 (approximately 6.3 kilobases), isolated from a rabbit uterus cDNA library, and PBRU-PCR33 (420 base pairs), produced by primer extension and PCR amplification. Compared with the chicken smooth muscle MHC sequence [Yanagisawa, M., Hamada, Y., Katsuragawa, Y., Imamura, M., Mikawa, T. & Masaki, T. (1987) J. Mol. Biol. 198, 143-157] the rabbit MHC shares about 90% amino acid identity in the S1 globular head region but shows a striking sequence divergence at the junction between the 25-kDa and 50-kDa proteolytic fragments of the functionally important S1 head domain. Genomic cloning shows that the rabbit smooth muscle MHC gene is large and has an unusual exon/intron organization at the 5' end. The first eight contiguous exons are located within a region of at least 70 kilobases of genomic DNA. Some introns span several kilobases of DNA and others at the 5' end show a high degree of intron conservation in the Mg(2+)-ATPase domain when compared with more distantly related sarcomeric MHC genes. Primer extension and S1 nuclease mapping analysis demonstrate that transcription initiates from a single site in the rabbit smooth muscle MHC gene.

Effects of corticosteroid on the transport and metabolism of glutamine in rat skeletal muscle.

Intramuscular glutamine falls with injury and disease in circumstances associated with increases in blood corticosteroids. We have investigated the effects of corticosteroid administration (0.44 mg/kg dexamethasone daily for 8 days, 200 g female rats) on intramuscular glutamine and Na+, muscle glutamine metabolism and sarcolemmal glutamine transport in the perfused hindlimb. After dexamethasone treatment intramuscular glutamine fell by 45% and Na+ rose by 25% (the respective muscle/plasma distribution ratios changed from 8.6 to 4.5 and 0.12 to 0.15); glutamine synthetase and glutaminase activities were unchanged at 475 +/- 75 and 60 +/- 19 nmol/g muscle per min. Glutamine output by the hindlimb of anaesthetized rats was increased from 31 to 85 nmol/g per min. Sarcolemmal glutamine transport was studied by paired-tracer dilution in the perfused hindlimb: the maximal capacity (Vmax) for glutamine transport into muscle (by Na(+)-glutamine symport) fell from 1058 +/- 310 to 395 +/- 110 nmol/g muscle per min after dexamethasone treatment, accompanied by a decrease in the Km (from 8.1 +/- 1.9 to 2.1 +/- 0.4 mM glutamine). At physiological plasma glutamine concentration (0.75 mM) dexamethasone appeared to cause a proportional increase in sarcolemmal glutamine efflux over influx. Addition of dexamethasone (200 nM) to the perfusate of control rat hindlimbs caused acute changes in Vmax and Km of glutamine transport similar to those resulting from 8-day dexamethasone treatment. The reduction in muscle glutamine concentration after dexamethasone treatment may be primarily due to a reduction in the driving force for intramuscular glutamine accumulation, i.e., in the Na+ electrochemical gradient. The prolonged increase in muscle glutamine output after dexamethasone treatment (which occurs despite a reduction in the size of the intramuscular glutamine pool) appears to be due to a combination of (a) accelerated sarcolemmal glutamine efflux and (b) increased intramuscular synthesis of glutamine.

Glutamine transport and metabolism in denervated rat skeletal muscle.

Rat skeletal muscle glutamine fell by 40% from 4.18 to 2.5 mumols/g wet weight (P less than 0.01) after 4 days of denervation. Over the same period net glutamine efflux from denervated hindlimbs [i.e., arteriovenous (a-v) concentration differences x blood flow] increased 3.5-fold (from -6.72 +/- 1.73 to -26 +/- 4.81 nmol.min-1.g-1, P less than 0.001). Gastrocnemius glutamine synthetase activity fell 48% after denervation (from 475 +/- 81 to 248 +/- 39 nmol.min-1.g-1, P less than 0.001), but glutaminase activity was not significantly altered (17 nmol.min-1.g-1). The maximal activity (Vmax) of the unidirectional Na(+)-dependent glutamine transporter (system Nm) was depressed by 45% from 1,020 +/- 104 to 571 +/- 9 nmol.min-1.g-1 (P less than 0.01), but the concentration at which transport was half maximal (Km) was not significantly altered (control 8.1 +/- 0.6 mM; denervated 6.52 +/- 0.12). Hindlimb denervation resulted in an increase of intramuscular Na+ by 17% and a fall of K+ by 12%, and the resting membrane potential in isolated muscles decreased from -75 +/- 10 to -59.5 +/- 5.5 mV. Membrane potential of perfused denervated muscle, isolated after acute addition of the Na+ channel blocker tetrodotoxin (TTX, 3 microM), repolarized to -66.4 +/- 3.2 mV. In perfused denervated preparations TTX caused an acute recovery of Vmax of unidirectional glutamine transport to 848 +/- 75 nmol.min-1.g-1; Km was unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)

Myosin heavy chain isoform diversity in smooth muscle is produced by differential RNA processing.

We have isolated and characterized two distinct myosin heavy chain cDNA clones from a neonatal rat aorta cDNA library. These clones encode part of the light meromyosin region and the carboxyl terminus of smooth muscle myosin heavy chain. The two rat aorta cDNA clones were identical in their 5' coding sequence but diverged at the 3' coding and in a portion of the 3' untranslated regions. One cDNA clone, RAMHC21, encoded 43 unique amino acids from the point of divergence of the two cDNAs. The second cDNA clone, RAMHC 15, encoded a shorter carboxyl terminus of nine unique amino acids and was the result of a 39 nucleotide insertion. This extra nucleotide sequence was not present in RAMHC21. The rest of the 3' untranslated sequences were common to both cDNA clones. Genomic cloning and DNA sequence analysis demonstrated that an exon specifying the 39 nucleotides unique to RAMHC15 mRNA was present, together with the 5' upstream common exons in the same contiguous stretch of genomic DNA. The 39 nucleotide exon is flanked on either side by two relatively large introns of approximately 2600 and 2700 bases in size. RNase protection analysis indicated that the two corresponding mRNAs were coexpressed in both vascular and non-vascular smooth muscle tissues. This is the first demonstration of alternative RNA processing in a vertebrate myosin heavy chain gene and provides a novel mechanism for generating myosin heavy chain protein diversity in smooth muscle tissues.

Identification of two types of smooth muscle myosin heavy chain isoforms by cDNA cloning and immunoblot analysis.

We previously reported the characterization of a rabbit uterus cDNA clone (SMHC29) which encoded part of the light meromyosin of smooth muscle myosin heavy chain (Nagai, R., Larson, D.M., and Periasamy, M. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 1047-1051). We have now characterized a second cDNA clone (SMHC40) which also encodes part of the light meromyosin but differs from SMHC29 in the following respects. Nucleotide sequence analysis demonstrates that the two myosin heavy chain mRNAs are identical over 1424 nucleotides but differ in part of the 3'-carboxyl coding region and a portion of the 3'-nontranslated sequence. Specifically, SMHC40 cDNA encodes a unique stretch of 43 amino acids at the carboxyl terminus, whereas SMHC29 cDNA contains a shorter carboxyl terminus of 9 unique amino acids which is the result of a 39-nucleotide insertion. Recent peptide mapping of smooth muscle myosin heavy chain identified two isotypes with differences in the light meromyosin fragment that were designated as SM1 (204 kDa) and SM2 (200 kDa) type myosin (Eddinger, T. J., and Murphy, R.A. (1988) Biochemistry 27, 3807-3811). In this study we present direct evidence that SMHC40 and SMHC29 mRNA encode the two smooth muscle myosin heavy chain isoforms, SM1 and SM2, respectively, by immunoblot analysis using antibodies against specific carboxyl terminus sequences deduced from SMHC40 and SMHC29 cDNA clones.

Sculpturing new muscle phenotypes.

Changes in the pattern of muscle activity are followed by new patterns of protein synthesis, both in the contractile elements and in the enzymes of energy metabolism. Although the signal transducers have not been identified, techniques of molecular biology have clearly shown that the adaptive responses are the regulated consequence of differential gene expression.

Alpha-actin and cytochrome c mRNAs in atrophied adult rat skeletal muscle.

Specific complementary DNA (cDNA) hybridization probes were used to estimate the levels of alpha-actin and cytochrome c mRNAs and also 18S rRNA in three models of skeletal muscle atrophy. After 7 days of hindlimb suspension, or immobilization, or denervation, protein content decreased 26-32% in all muscles studied except suspended fast-twitch muscle, which lost only half as much protein. alpha-Actin mRNA content decreased 51-66% and cytochrome c mRNA content decreased 42-61% in slow- and fast-twitch muscles in all three models of atrophy. However, total RNA content did not show similar directional changes; RNA content decreased 27-44% in suspended and immobilized muscle but was unchanged in denervated fast-twitch muscle. The results were interpreted to suggest that loss of weight-bearing function of skeletal muscle is a major factor affecting the levels of alpha-actin and cytochrome c mRNAs during muscle atrophy.

Clenbuterol prevents or inhibits loss of specific mRNAs in atrophying rat skeletal muscle.

It is known that denervation or hindlimb suspension both decrease the content of rRNA, alpha-actin mRNA, and cytochrome c mRNA in adult rat skeletal muscle. In the present study, the provision of clenbuterol (an anabolic agent) to adult female rats during a 7-day period of denervation of the soleus and gastrocnemius muscles prevented entirely the loss of rRNA, alpha-actin mRNA, and cytochrome c mRNA that normally occurs in denervated muscle. Although clenbuterol inhibited most of the loss of alpha-actin mRNA that occurred in the soleus and gastrocnemius muscles after 7 days of hindlimb suspension, clenbuterol administration had less effect on preventing the loss of rRNA and cytochrome c mRNA in hindlimb suspended skeletal muscle. Clenbuterol had no effect on protein content in atrophied muscle resulting from denervation or suspension. These data suggest that clenbuterol can maintain the expression of certain RNAs in atrophying adult rat skeletal muscle.

Adaptation of muscle gene expression to changes in contractile activity.

NASA: A review of the existing literature regarding the effects of different types of physical activities on the gene expression of adult skeletal muscles leads us to conclude that each type of exercise training program has, as a result, a different phenotype, which means that there are multiple mechanisms, each producing a unique phenotype. A portion of the facts which support this position is presented and interpreted here. [Abstract translated from the original French by NASA]^ieng

Characteristics of a glutamine carrier in skeletal muscle have important consequences for nitrogen loss in injury, infection, and chronic disease.

A carrier for glutamine, identified in rat muscle, has properties in terms of kinetics, ion dependence and hormone sensitivity, and effects of endotoxin and branched-chain aminoacids that point to an important function in the control of whole-body aminoacid metabolism. The existence of a link between the size of the glutamine pool in muscle and the rate of muscle protein synthesis raises possibilities for therapeutic interventions to limit protein loss in injury, sepsis, and chronic disease.

Changes in blood ammonia, lactate and amino acids in relation to workload during bicycle ergometer exercise in man.

Whole body exercise at intensities up top 50% VO2max has no effect on the concentration of blood ammonia but a threefold rise in blood ammonia is observed at workloads up to maximal. There is a linear relationship between blood ammonia and lactate production during exercise which suggests that the two processes may be linked to a common process of short-term energy provision. Blood glutamine and blood alanine both show rises linearly related to power output during exercise, suggesting that if these amino acids are sinks for ammonia then the process of ammonia incorporation is saturated at high workloads.