Changes In Muscle Phenotype Research Articles

Myosin heavy chain isoform mRNA and protein levels after long-term paralysis

To assess the long-term influence of paralysis on muscle phenotypic mRNA and protein expression, the effects of spinal cord transection (ST) on myosin heavy chain (MyHC) isoform mRNA and protein levels in the soleus and medial gastrocnemius (MG) muscles of rats were analyzed. Control soleus contained predominantly MyHC-I with low amounts of MyHC-IIa and IIx mRNAs. After ST, MyHC-I mRNA decreased to ∼15%, MyHC-IIa was increased by 75–200%, and MyHC-IIx was elevated by 8–10×. Low level expression of MyHC-IIb was observed post-ST, suggesting that reduced activity is not a primary stimulus for MyHC-IIb expression. Adaptations in mRNA preceded protein adaptations in the soleus. Although MyHC-I protein in the MG was reduced post-ST, no other consistent changes occurred. The relative lack of adaptation to ST by the MG suggests that the reduced activity and load bearing encountered by the MG were insufficient to induce a change in muscle phenotype.

Regulation of Egr-1, SRF, and Sp1 mRNA expression in contracting skeletal muscle cells.

The early cellular signals associated with contractile activity initiate the activation and induction of transcription factors that regulate changes in skeletal muscle phenotype. The transcription factors Egr-1, Sp1, and serum response factor (SRF) are potentially important mediators of mitochondrial biogenesis based on the prevalence of binding sites for them in the promoter regions of genes encoding mitochondrial proteins, including PGC-1 alpha, the important regulator of mitochondrial biogenesis. Thus, to further define a role for transcription factors at the onset of contractile activity, we examined the time-dependent alterations in Egr-1, Sp1, and SRF mRNA and the levels in electrically stimulated mouse C(2)C(12) skeletal muscle cells. Early transient increases in Egr-1 mRNA levels within 30 min (P < 0.05) of contractile activity led to threefold increases (P < 0.05) in Egr-1 protein by 60 min. The increase in Egr-1 mRNA was not because of increased stability, as Egr-1 mRNA half-life after 30 min of stimulation showed only a 58% decline. Stimulation of muscle cells had no effect on Sp1 mRNA but led to progressive increases (P < 0.05) in SRF mRNA by 30 and 60 min. This was not matched by increases in SRF protein but occurred coincident with increases (P < 0.05) in SRF-serum response element DNA binding at 30 and 60 min as a result of SRF phosphorylation on serine-103. To assess the importance of the recovery period, 12 h of continuous contractile activity was compared with four successive 3-h bouts, with an intervening 21-h recovery period after each bout. Continuous contractile activity led to a twofold increase (P < 0.05) in Egr-1 mRNA, no change in SRF mRNA, and a 43% decrease in Sp1 mRNA expression. The recovery period prevented the decline in Sp1 mRNA, produced a decrease in Egr-1 mRNA, and had no effect on SRF mRNA. Thus continuous and intermittent contractile activity evoked different specific transcription factor expression patterns, which may ultimately contribute to divergent qualitative, or temporal patterns of, phenotypic adaptation in muscle.

Muscle molecular phenotype after stroke is associated with gait speed.

The disability of patients after stroke is generally attributed to upper motor neuron defects, but secondary changes in paretic muscle may enhance the disability. We analyzed the molecular phenotype and metabolic profile of the paretic and nonparetic vastus lateralis (VL) and we measured the severity of gait deficit in 13 patients at least 6 months after ischemic stroke. The results showed a significant increase in the proportion of fast myosin heavy chain (MHC, 68 +/- 14%) in the paretic compared to the nonparetic VL (50 +/- 13%). The specific activity of citrate synthase and glyceraldehyde phosphodehydrogenase was not significantly different between the two sides. The proportion of fast MHC was inversely associated with severity of gait deficit indexed by self-selected walking speed in the paretic leg, but not the nonparetic leg. Our findings demonstrate significant and potentially modifiable secondary biologic changes in hemiparetic muscle phenotype that may contribute to the disability of stroke.

Effect of Chronic Muscle Denervation on Mitochondrial Biogenesis and Apoptosis

1825 Chronic muscle disuse, such as that produced by denervation, reduces mitochondrial content and produces muscle atrophy. These changes in muscle phenotype could be provoked by alterations in important regulators of mitochondrial biogenesis, such as the nuclear coactivator PGC-1α, and by increases in muscle fiber cell death via apoptosis. However, the role of PGC-1α following denervation has not been defined, and limited research has assessed apoptotic mechanisms as contributing factors to denervation-induced muscle atrophy. PURPOSE: To assess time-dependent alterations in key proteins involved in mitochondrial biogenesis (PGC-1α, cytochrome c, Tfam, cytochrome oxidase (COX) activity) and apoptosis (HSP70, Bcl-2, BAX, AIF) following chronic muscle denervation. METHODS: Male Sprague-Dawley rats (n = 20) underwent unilateral denervation (DEN) of the sciatic nerve for 5, 21, or 42 days (n = 3–4 animals/day). Denervated and contralateral (control) red gastrocnemius muscles were used for biochemical analyses. RESULTS: Denervation decreased PGC-1α expression by 45% following 5 days of DEN, which was further decreased by 51% and 65% of control at 21 and 42 days, respectively. COX activity declined with time and matched PGC-1α expression, with decreases of 35%, 55%, and 70% at 5, 21, and 42 days of DEN. These changes occurred coincident with 45% decreases in both Tfam and cytochrome c expression at 42 days of DEN. In contrast, proapoptotic BAX expression was elevated by 88% and 115% after 21 and 42 days of DEN, while coincident decreases of 79% and 89% occurred for the expression of the antiapoptotic protein Bcl-2. These changes dramatically increased the BAX:Bcl-2 ratio in denervated muscle. An increase in this ratio is indicative of a greater susceptibility to apoptosis. Additionally, 42 days of DEN resulted in increased (2.0-fold) expression of pro-apoptotic AIF, as well as increased levels (2.9-fold) of anti-apoptotic HSP70. CONCLUSIONS: These results indicate PGC-1α could be an important mediator of the decrements in mitochondrial content following periods of chronic muscle disuse. In addition, the profound shift in BAX:Bcl-2 ratio suggests that denervated muscle has a greater susceptibility to apoptosis and that this likely contributes to denervationinduced atrophy of skeletal muscle. Support: NSERC, Canada.

Constitutive expression of inducible Hsp70 is linked to natural shifts in skeletal muscle phenotype.

Constitutive expression of heat shock protein 70 (Hsp70) is elevated in frequently recruited, metabolically efficient rodent striated muscle. We aimed to assess the relative importance of muscle phenotype vs. increased contractile activity on this pattern of expression using the rat diaphragm, which undergoes a dramatic and sustained increase in recruitment with parturition and development. Diaphragms were collected from rats of various ages (20 day fetus, 1 and 3 days, and 1, 3, 6 and 12 weeks postpartum; PP), and assessed for changes in oxidative capacity, Hsp70 and Type I myosin heavy chain (MHCI) (used as a marker of muscle phenotype changes). Oxidative capacity of the diaphragm (as indicated by citrate synthase activity) and whole body growth rate (% increase in body weight per week), factors thought to require chaperone activity, increased rapidly, peaked at 3-6 weeks PP and declined late in development. In contrast, at 1 week PP, increased contractile activity in the diaphragm had not altered the expression of Hsp70 protein or mRNA from fetal levels. Significant increases in Hsp70 were not observed until between 1 and 3 weeks, achieving their highest levels at 12 weeks PP. Both MHC I protein (r = 0.69, P = 0.001) and mRNA (r = 0.76, P = 0.001) were significantly correlated with their Hsp70 counterparts. Expression of Hsp70 in the developing diaphragm represents an adaptation associated with a shift towards a slower, more metabolically efficient adult phenotype rather than simply a response to contractile stress.

The MEF2 site is necessary for induction of the myosin light chain 2 slow promoter in overloaded regenerating plantaris muscle

Functional overload (OV) of the rat plantaris muscle results in a fast to slow change in muscle phenotype with induction of the slow contractile protein genes including myosin light chain 2 slow (MLC2s). To identify potential cis-acting DNA sites regulating MLC2s following ablation, plasmid constructs were transfected in vivo into regenerating overloaded plantaris muscles. Activity of the 270bp promoter (−270MLC2s) was increased in OV muscles at 28 days. Mutation of the MEF2 site (−270MEF2) knocked out the overload-induced activity of the promoter. Mutation of the Ebox (−270Ebox) resulted in an earlier induction with OV and mutation of the CACC site (–270CACC) resulted in increased activity in the CON PLN with OV induction detected by 21 days. These results demonstrate that the –270MLC2s promoter contains the elements necessary for expression of MLC2s in regenerating OV PLN. More importantly, mutation analysis of −270MLC2s promoter demonstrates that mechanical loading induced expression shares some common molecular mechanisms with slow nerve dependent model regulation. In these two models of physiological induction of MLC2s, the CACC site acts as a repressor region (on/off switch) and the MEF2 site acts to modulate quantitative expression.

Myosin heavy chain expression and atrophy in rat skeletal muscle during transition from cardiac hypertrophy to heart failure.

The purpose of this investigation was to determine whether changes in myosin heavy chain (MHC) expression and atrophy in rat skeletal muscle are observed during transition from cardiac hypertrophy to chronic heart failure (CHF) induced by aortic stenosis (AS). AS and control animals were studied 12 and 18 weeks after surgery and when overt CHF had developed in AS animals, 28 weeks after the surgery. The following parameters were studied in the soleus muscle: muscle atrophy index (soleus weight/body weight), muscle fibre diameter and frequency and MHC expression. AS animals presented decreases in both MHC1 and type I fibres and increases in both MHC2a and type IIa fibres during late cardiac hypertrophy and CHF. Type IIa fibre atrophy occurred during CHF. In conclusion, our data demonstrate that skeletal muscle phenotype changes occur in both late cardiac hypertrophy and heart failure; this suggests that attention should be given to the fact that skeletal muscle phenotype changes occur prior to overt heart failure symptoms.

AT1 receptor antagonist inhibits aortic smooth muscle phenotypic change through no and AKT signaling in hypertension in vivo

The precise mechanisms of angiotensin II type 1 (AT1) receptors on the vascular smooth muscle cell (VSMCs) phenotype in vivo remain unclear, as studies using passaged VSMCs under conventional culture conditions have been unable to adequately investigate the molecular mechanisms underlying the phenotypic modulation of VSMCs. To clarify the mechanisms of smooth muscle (SM) cell phenotype and AT1 receptor-mediated signal transductions in hypertension, we compared the effects of an angiotensin-converting enzyme (ACE) inhibitor and AT1 receptor antagonist on vascular remodeling, oxidative stress and phenotypic changes of VSMCs. Fourteen-week-old male stroke-prone spontaneously hypertensive rats (SHRSP) were divided into 3 groups and treated for 6 weeks with either a vehicle, an ACE inhibitor (cilazapril; 10 mg/kg/day), and AT1 receptor antagonist (E4177; 30 mg/kg/day). Compared to the vehicle group, blood pressure, wall-to-lumen ratio, collagen content and the total cell number in aortic media were significantly reduced in the 2 drug-treated groups; with no significant differences between the 2 drug-treated groups. Both drugs significantly up-regulated the contractile-type SM myosin heavy chain (MHC); SM2. Whereas, they significantly down-regulated the synthetic-type SM-MHC; NMHC-B/SMemb. Total aortic reactive oxygen species (ROS) assessed by 8-iso-PGF2α, the expression of NAD(P)H oxidase essential subunit, p22phox, and p38 mitogen-activated protein kinase (p38 MAPK) were significantly reduced in the 2 drug-treated groups, and superoxide content of the aortic media assessed by hydroxyetidine staining did not differ between the 2 drug-treated groups. However, eNOS, phosphoinositide 3-kinase (PI3-K)/protein kinase B (Akt) and Cu/Zn SOD were significantly up-regulated in the 2 drug-treated groups. Additionally, E4177 induced greater increases in SM2, eNOS and p-Akt expression than did cilazapril. AT1 receptor antagonists inhibited aortic VSMC phenotypic changes by the up-regulation of eNOS expression and activation of the Akt pathway, but not p38-MAPK signaling, than did ACE inhibitors in SHRSP; suggesting that AT1 receptor-mediated NO and Akt signaling might be an important determinant pathway for the aortic VSMC phenotype in hypertension in vivo.

Exercise, diet, and skeletal muscle gene expression.

Skeletal muscle, as a consequence of its mass and great capacity for altered metabolism, has a major impact on whole-body metabolic homeostasis and is capable of remarkable adaptation in response to various physiological stimuli, including exercise and dietary intervention. Exercise-induced increases in skeletal muscle mRNA levels of a number of genes have been reported, due to transcriptional activation and/or increased mRNA stability. The cellular adaptations to exercise training appear to be due to the cumulative effects of transient increases in gene transcription after repeated exercise bouts. The relative importance of transcriptional (mRNA synthesis) and translational (mRNA stability or translational efficiency) mechanisms for the training-induced increases in skeletal muscle protein abundance remains to be fully elucidated. Dietary manipulation, and the associated alterations in nutrient availability and hormone levels, can also modify skeletal muscle gene expression, although fewer studies have been reported. A major challenge is to understand how exercise and diet exert their effects on gene and protein expression in skeletal muscle. In relation to exercise, potential stimuli include stretch and muscle tension, the pattern of motor nerve activity and the resultant calcium transients, the energy charge of the cell and substrate availability, oxygen tension and circulating hormones. These are detected by various cellular signaling mechanisms, acting on a range of downstream targets and a wide range of putative transcription factors. A key goal in the years ahead is to identify how alterations at the level of gene expression are coupled to the changes in skeletal muscle phenotype. It is clear that gene expression, although representing a specific site of regulation, is only one step in a complex cascade from the initial stimulus to the final phenotypic adaptation and integrated physiological response.

Molecular Basis by Which Garlic Suppresses Atherosclerosis

The aim of this study was to determine the mechanism by which the aged garlic extract "Kyolic" has a protective effect against atherosclerosis. Plasma cholesterol of rabbits fed a 1% cholesterol-enriched diet for 6 wk was not reduced by supplementation with 800 microL Kyolic/(kg body. d). In spite of this, Kyolic reduced by 64% (P < 0.05) the surface area of the thoracic aorta covered by fatty streaks and significantly reduced aortic arch cholesterol. Kyolic also significantly inhibited by approximately 50% the development of thickened, lipid-filled lesions in preformed neointimas produced by Fogarty 2F balloon catheter injury of the right carotid artery in cholesterol-fed rabbits. In vitro studies found that Kyolic completely prevented vascular smooth muscle phenotypic change from the contractile, high volume fraction of filament (V(v)myo) state, and inhibited proliferation of smooth muscle cells in the synthetic state with a 50% effective dose (ED(50)) of 0.2%. Kyolic also slightly inhibited the accumulation of lipid in cultured macrophages but not smooth muscle, and had no effect on the expression of adhesion molecules on the surface of the endothelium or the adherence of leukocytes. It is concluded that Kyolic exerts antiatherogenic effects through inhibition of smooth muscle phenotypic change and proliferation, and by another (unclarified) effect on lipid accumulation in the artery wall.

Cis-acting DNA Elements of Mouse Granulocyte/Macrophage Colony-stimulating Factor Gene Responsive to Oxidized Low Density Lipoprotein

We previously demonstrated that the induction of granulocyte/macrophage colony-stimulating factor (GM-CSF) played an important role in oxidized low density lipoprotein (Ox-LDL)-induced macrophage growth as a growth priming factor. The present study was undertaken to elucidate the transcriptional regulation of the GM-CSF gene using Raw 264.7 cells, a mouse macrophage cell line. Transient transfection into Raw 264.7 cells of several 5'-flanking regions of GM-CSF gene-luciferase fusion plasmids revealed the presence of two positive regulatory sites in regions spanning from -97 to -59 and from -59 to -37 and one negative regulatory site from -120 to -97 in unstimulated cells. When cells were stimulated by Ox-LDL, there was one positive responsive site from -225 to -120 and one negative responsive site from -97 to -59, which contained the NF-kappaB binding site. Computer analysis revealed the presence of a putative AP-2 binding site from -169 to -160. Mutagenesis of a putative AP-2 binding site and tandem repeat of this site in plasmid resulted in a complete loss and increased responsiveness to Ox-LDL, respectively. Electrophoretic mobility shift assay showed that Ox-LDL increased the binding of certain nuclear protein(s) to a putative AP-2 binding site but decreased their binding to NF-kappaB binding site. Supershift assay showed that nuclear proteins bound to NF-kappaB binding site contained, at least, p50 and p65 but could not demonstrate nuclear protein(s) bound to a putative AP-2 binding site. Our results suggested that a putative AP-2 binding site from -169 to -160 was a positive responsive element to Ox-LDL and that the NF-kappaB binding site from -91 to -82 was a negative responsive element in Ox-LDL-induced GM-CSF transcription.

Changes in muscle mass and phenotype and the expression of autocrine and systemic growth factors by muscle in response to stretch and overload.

The study of the underlying mechanisms by which cells respond to mechanical stimuli, i.e. the link between the mechanical stimulus and gene expression, represents a new and important area in the morphological sciences. Several cell types ('mechanocytes'), e.g. osteoblasts and fibroblasts as well as smooth, cardiac and skeletal muscle cells are activated by mechanical strain and there is now mounting evidence that this involves the cytoskeleton. Muscle offers one of the best opportunities for studying this type of mechanotransduction as the mechanical activity generated by and imposed upon muscle tissue can be accurately controlled and measured in both in vitro and in vivo systems. Muscle is highly responsive to changes in functional demands. Overload leads to hypertrophy, whilst decreased load force generation and immobilisation with the muscle in the shortened position leads to atrophy. For instance it has been shown that stretch is an important mechanical signal for the production of more actin and myosin filaments and the addition of new sarcomeres in series and in parallel. This is preceded by upregulation of transcription of the appropriate genes some of which such as the myosin isoforms markedly change the muscle phenotype. Indeed, the switch in the expression induced by mechanical activity of myosin heavy chain genes which encode different molecular motors is a means via which the tissue adapts to a given type of physical activity. As far as increase in mass is concerned, our group have cloned the cDNA of a splice variant of IGF-1 that is produced by active muscle that appears to be the factor that controls local tissue repair, maintenance and remodelling. From its sequence it can be seen that it is derived from the IGF-1 gene by alternative splicing but it has different exons to the liver isoforms. It has a 52 base insert in the E domain which alters the reading frame of the 3' end. Therefore, this splice variant of IGF-1 is likely to bind to a different binding protein which exists in the interstitial tissue spaces of muscle, neuronal tissue and bone. This would be expected to localise its action as it would be unstable in the unbound form which is important as its production would not disturb the glucose homeostasis unduly. This new growth factor has been called mechano growth factor (MGF) to distinguish it from the liver IGFs which have a systemic mode of action. Although the liver is usually thought of as the source of circulating IGF-1, it has recently been shown that during exercise skeletal muscle not only produces much of the circulating IGF-1 but active musculature also utilises most of the IGF-I produced. We have cloned both an autocrine and endocrine IGF-1, both of which are upregulated in cardiac as well as skeletal muscle when subjected to overload. It has been shown that, in contrast to normal muscle, MGF is not detectable in dystrophic mdx muscles even when subjected to stretch and stretch combined with electrical stimulation. This is true for muscular dystrophies that are due to the lack of dystrophin (X-linked) and due to a laminin deficiency (autosomal), thus indicating that the dystrophin cytoskeletal complex may be involved in the mechanotransduction mechanism. When this complex is defective the necessary systemic as well as autocrine IGF-1 growth factors required for local repair are not produced and the ensuing cell death results in progressive loss of muscle mass. The discovery of the locally produced IGF-1 appears to provide the link between the mechanical stimulus and the activation of gene expression.

Development of nicotinic receptor clusters and innervation accompanying the change in muscle phenotype in the mouse esophagus.

During development, the external muscle of the mouse esophagus undergoes a transdifferentiation from smooth to striated muscle (Patapoutian et al. [1995] Science 270:1818-1821). We now report on the development of the innervation accompanying the change in phenotype of the external muscle of the mouse esophagus. The phenotype of the muscle was monitored by using light and electron microscopy. Nicotinic acetylcholine receptors were localised by using a fluorescence conjugate of alpha-bungarotoxin, and neural elements were localised by using antisera to synaptophysin (a synaptic vesicle protein that was used to label all nerve terminals), the vesicular acetylcholine transporter (VAChT), calcitonin gene-related peptide (CGRP), nitric oxide synthase (NOS), and vasoactive intestinal peptide (VIP). CGRP and VAChT were co-localised in the terminals of vagal motoneurons that innervate the external muscle, and NOS and VIP were co-localised in intrinsic (enteric) neurons, which provide some terminals that are associated with motor endplates. Cells exhibiting striations were first observed in the outer layers of the most rostral regions of the esophagus of embryonic day 15 (E15) mice. Clusters of nicotinic acetylcholine receptors were also first observed at the rostral end of the esophagus of E15 mice, and developed in a rostrocaudal progression that coincided with the appearance of striations within the muscle cells. Synaptophysin-, VAChT- and NOS-immunoreactive nerve terminals were present within the external muscle prior to the formation of receptor clusters, and their appearance did not follow any apparent rostrocaudal sequence. Surprisingly, not all of the receptor clusters at E15 had synaptophysin- and VAChT-immunoreactive nerve terminals closely associated with them. However, from E18 on, almost all of the clusters had synaptophysin-immunoreactive nerve terminals in close association. At late embryonic and early postnatal stages, there was a rostrocaudal gradient in the proportion of receptor clusters having VAChT-immunoreactive nerve terminals associated with them. Nerve terminals associated with nicotinic receptor clusters did not show detectable CGRP-immunoreactivity until one to two weeks after the appearance of synaptophysin- and VAChT-immunoreactivity. The NOS-immunoreactive neurons did not show detectable VIP-immunoreactivity until three days after NOS could be detected. These results show that the appearance of clusters of nicotinic receptors in the external muscle of the esophagus coincides with the expression of a striated muscle phenotype, but not with the presence of ingrowing nerve terminals. However, many of the receptor clusters that were observed first were apparently uninnervated.

Expression of MyoD in Cultured Primary Myotubes Is Dependent on Contractile Activity: Correlation with Phenotype-Specific Expression of a Sarcoplasmic Reticulum Ca2+-ATPase Isoform

Myogenic determination factors (MDF) have been implicated in the establishment and maintenance of the fast or slow phenotype in skeletal muscle, with MyoD favoring the fast and myogenin favoring the slow phenotype. Accordingly, contractility-induced changes in muscle phenotype should be accompanied by a change in the MyoD/myogenin ratio. Some reports show such changes, but limitations inherent toin vivostudies complicate interpretation of these data. Here we tested whether a relationship can be found between contractility, MDF expression, and the expression of phenotype-specific muscle proteins in a simplein vitrosystem of cultured primary myotubes. We show that contractions reduce the MyoD/myogenin ratio by specifically repressing MyoD mRNA expression. This is accompanied by a selective repression at a pretranslational level of the expression of fast-type sarcoplasmic reticulum Ca2+-ATPase. Thesein vitroresults support a phenotype-determining role of MDFs as a function of contractile activity and show that cultured myotubes can be a useful model for the analysis of the molecular mechanism of such regulation of muscle phenotype.

Changes in adult muscle phenotype in response to disuse and passive stretch.

Changes in adult muscle phenotype in response to disuse and passive stretch.

Heparan sulfate-degrading enzymes induce modulation of smooth muscle phenotype

Macrophages cocultured with rabbit aortic smooth muscle cells at a ratio of 1:3 degraded all the 35S-labeled heparan sulfate proteoglycan from the smooth muscle surface into free sulfate (Kav of 0.84 on Sepharose 6B). Concomitantly, the same macrophages induced a decrease in the volume fraction of myofilaments (Vvmyo) of the smooth muscle cells and a decrease in alpha-actin mRNA as a percentage of total actin mRNA. Both macrophage lysosomal lysate at neutral pH and heparinase degraded cell-free 35S-labeled matrix deposited by smooth muscle cells into fragments which eluted at a Kav of 0.63 and which were identified as heparan sulfate chains by their complete degradation in the presence of low pH nitrous acid. At acid pH the macrophage lysosomal lysate completely degraded the heparan sulfate to free sulfate (Kav 0.84). Both macrophage lysosomal lysate and commercial heparinase at neutral pH induced smooth muscle phenotypic change while other enzymes such as trypsin and chondroitin ABC lyase had no effect. It was therefore suggested that the active factor present in the macrophages is a lysosomal heparan sulfate-degrading endoglycosidase (heparinase). Only a small amount of heparan sulfate-degrading activity was released into the incubation medium by living macrophages, and there was no heparinase activity on their isolated plasma membranes, although proteolytic enzymes were evident in both instances. In pulse-chase studies, high Vvmyo smooth muscle cells were seen to constantly internalize and degrade 35S-labeled heparan sulfate proteoglycan from their own pericellular compartment, suggesting that this may be the mechanism by which smooth muscle phenotype is maintained under normal circumstances and that removal of heparan sulfate from the surface of smooth muscle cells and its degradation by macrophages temporarily interrupts this process, inducing smooth muscle phenotypic change.

Molecular mechanisms of skeletal muscle adaptations to exercise

The strength and oxidative capacity of skeletal muscle are major determinants of physical performance, whether in elite athletes or in patients with congestive heart failure or other circulatory disorders. Exercise training augments the functional capacity of skeletal muscle, in part by altering the abundance of proteins essential for contraction and energy metabolism. These adaptations result from exercise-induced changes in the abundance of specific mRNA transcripts, suggesting that gene transcription is an important target for signaling pathways that couple contractile activity to changes in muscle phenotype. Understanding the genetic control mechanisms involved in the adaptive capacity of skeletal muscle could lead to novel approaches to the therapy of patients with cardiovascular disease, either by improving peripheral utilization of oxygen in patients with limited cardiac output or by contributing to the successful application of skeletal muscle grafts to assist the failing heart.

THE MACROPHAGE AS AN INITIATOR OF ATHEROSCLEROSIS

1. Heparan sulfate proteoglycan in the basal lamina of smooth muscle cells is important in the maintenance of the 'contractile', high volume fraction of myofilaments (Vvmyo) phenotype. The mechanism by which this occurs may involve the continuous internalization of heparan sulfate by the smooth muscle cells themselves. 2. One macrophage can degrade all the heparan sulfate from three smooth muscle cells by the action of heparan sulfate-degrading enzymes in their lysosomes, thus leaving none available for internalization by the smooth muscle cell until it has synthesized more, and leading to the induction of smooth muscle phenotypic change from a high Vvmyo to a low Vvmyo. 3. In this altered phenotype the smooth muscle cells proliferate in response to mitogens, synthesize large amounts of extracellular matrix and accumulate lipid, all characteristics of the smooth muscle cell in developing atheroma.

Smooth muscle phenotypic changes in arterial wall homeostasis: Implications for the pathogenesis of atherosclerosis

Smooth muscle phenotypic changes in arterial wall homeostasis: Implications for the pathogenesis of atherosclerosis

Lipid accumulation in arterial smooth muscle cells: Influence of phenotype

Isolated smooth muscle cells from the adult pig and rabbit aorta in primary culture undergo a spontaneous change in phenotype from a contractile to a synthetic state over 6–8 days, losing their capacity to contract and gaining the capacity to divide. The change in smooth muscle phenotype to the synthetic state is accompanied by distinct changes in the cells' ability to metabolize LDL, with the rate of degradation of 125I-labelled LDL decreasing to about one fifth of the level in contractile state cells. This does not appear to be due to changes in the number or affinity of LDL receptors since saturable binding of LDL is unaltered. The specific activities of the lysosomal enzymes acid phosphatase and N-acetyl-β-glucosamini-dase increase with change to the synthetic state as do cytochrome c oxidase (mitochondria) and NADPH-dependent cytochrome c reductase (endoplasmic reticulum). In contrast there is a slight but not significant decrease in the specific activity of the lysosomal enzyme acid cholesteryl esterase of rabbit smooth muscle cells and a significant decrease in the activity of pig cells with change in phenotype to the synthetic state. Significantly more [ 3H]cholesteryl oleate is recovered in synthetic state than in contractile state cells following incubation with 20 tug/ml unlabelled LDL and [ 3H]sodium oleate. Morphologically there is no difference in the number of lipid droplets in contractile and synthetic state cells after incubation in 5% normolipemic serum, but in cells grown in 10% hyperlipemic serum for 4 days synthetic state cells become almost completely filled with lipid droplets while contractile state cells are unaffected. Lipid accumulation also occurs selectively in vivo in synthetic is compared with contractile state smooth muscle cells within intimal fibromuscular thickening induced by de-endothelialization of the carotid artery of cholesterol-fed rabbits. We suggest that accumulation of lipid in smooth muscles cells of atherosclerotic plaques is related to reduced catabolism of LDL following smooth muscle phenotypic change from the contractile to synthetic state.