Endothelial nitric oxide synthase induces heat shock protein HSPA6 (HSP70B') in human arterial smooth muscle cells.

Endothelial nitric oxide synthase (eNOS) is the major source of nitric oxide (NO) production in blood vessels. One of the pleitropic functions of eNOS derived NO is to inhibit vascular smooth muscle cell proliferation in the blood vessel wall, and whose dysfunction is a primary cause of atherosclerosis and restenosis. In this study there was an interest in examining the gene profile of eNOS adenoviral (Ad-eNOS) transduced human coronary artery smooth muscle cells (HCASMC) to further understand the eNOS inhibitory effect on smooth muscle cell proliferation. To this aim a whole genome wide analysis of eNOS transduced HCASMCs was performed. A total of 19 genes were up regulated, and 31 genes down regulated in Ad-eNOS transduced HCASMCs compared to cells treated with an empty adenovirus. Noticeably, a cluster of HSP70 gene family members was amongst the genes up regulated. Quantitative PCR confirmed that transcripts for HSPA1A (HSP70A), HSPA1B (HSP70B) and HSPA6 (HSP70B') were elevated 2, 1.7 and 14-fold respectively in Ad-eNOS treated cells. The novel gene HSPA6 was further explored as a potential mediator of eNOS signaling in HCASMC. Immunoblotting showed that HSPA6 protein was induced by Ade-NOS. To functionally examine the effect of HSPA6 on SMCs, an adenovirus harboring the HSPA6 gene under the control of a constitutive promoter was generated. Transduction of HCASMCs with Ad-HSPA6 inhibited SMC proliferation at 3 and 6 days post serum growth stimulation, and paralleled the Ad-eNOS inhibition of SMC growth. The identification in this study that HSPA6 overexpression inhibits SMC proliferation coupled with the recent finding that inhibition of HSP90 has a similar effect, progresses the field of targeting HSPs for vascular repair.

Gene-eluting stents: non-viral, liposome-based gene delivery of eNOS to the blood vessel wall in vivo results in enhanced endothelialization but does not reduce restenosis in a hypercholesterolemic model.

Although successful, drug-eluting stents require significant periods of dual anti-platelet therapy with a persistent risk of late stent thrombosis due to inhibition of re-endothelialization. Endothelial regeneration is desirable to protect against in-stent thrombosis. Gene-eluting stents may be an alternative allowing inhibition of neointima and regenerating endothelium. We have shown that adenoviral endothelial nitric oxide synthase (eNOS) delivery can result in significantly decreased neointimal formation and enhanced re-endothelialization. Here, we examined non-viral reporter and therapeutic gene delivery from a stent. We coated lipoplexes directly onto the surface of stents. These lipostents were then deployed in the injured external iliac artery of either normal or hypercholesterolemic New Zealand White rabbits and recovered after 28 days. Lipoplexes composed of lipofectin and a reporter lacZ gene or therapeutic eNOS gene were used. We demonstrated efficient gene delivery at 28 days post-deployment in the media (21.3±7.5%) and neointima (26.8±11.2%). Liposomal delivery resulted in expression in macrophages between the stent struts. This resulted in improved re-endothelialization as detected by two independent measures compared with vector and stent controls (P<0.05 for both). However, in contrast to viral delivery of eNOS, liposomal eNOS does not reduce restenosis rates. The differing cell populations targeted by lipoplexes compared with adenoviral vectors may explain their ability to enhance re-endothelialization without affecting restenosis. Liposome-mediated gene delivery can result in prolonged and localized transgene expression in the blood vessel wall in vivo. Furthermore, lipoeNOS delivery to the blood vessel wall results in accelerated re-endothelialization; however, it does not reduce neointimal formation.

Utrophin upregulation in Duchenne muscular dystrophy.

Duchenne Muscular Dystrophy (DMD) is a devastating, progressive muscle wasting disease for which there is currently no effective treatment. DMD is caused by mutations in the dystrophin gene many of which result in the absence of the large cytoskeletal protein dystrophin at the sarcolemma. Over-expression of utrophin, the autosomal paralogue of dystrophin, as a transgene in the mdx mouse (the mouse model of DMD) has demonstrated that utrophin can prevent the muscle pathology. Thus, up-regulation of utrophin in DMD muscle is a potential therapy for DMD. In this review we discuss recent advances in our understanding of the regulatory pathways controlling utrophin expression and the various approaches that have been applied to increasing the level of utrophin in the mdx mouse. These results are very encouraging and suggest that pharmacological up-regulation of utrophin may well be a feasible approach to therapy for DMD.

IGF-1 induces skeletal myocyte hypertrophy through calcineurin in association with GATA-2 and NF-ATc1.

Localized synthesis of insulin-like growth factors (IGFs) has been broadly implicated in skeletal muscle growth, hypertrophy and regeneration. Virally delivered IGF-1 genes induce local skeletal muscle hypertrophy and attenuate age-related skeletal muscle atrophy, restoring and improving muscle mass and strength in mice. Here we show that the molecular pathways underlying the hypertrophic action of IGF-1 in skeletal muscle are similar to those responsible for cardiac hypertrophy. Transfected IGF-1 gene expression in postmitotic skeletal myocytes activates calcineurin-mediated calcium signalling by inducing calcineurin transcripts and nuclear localization of calcineurin protein. Expression of activated calcineurin mimics the effects of IGF-1, whereas expression of a dominant-negative calcineurin mutant or addition of cyclosporin, a calcineurin inhibitor, represses myocyte differentiation and hypertrophy. Either IGF-1 or activated calcineurin induces expression of the transcription factor GATA-2, which accumulates in a subset of myocyte nuclei, where it associates with calcineurin and a specific dephosphorylated isoform of the transcription factor NF-ATc1. Thus, IGF-1 induces calcineurin-mediated signalling and activation of GATA-2, a marker of skeletal muscle hypertrophy, which cooperates with selected NF-ATc isoforms to activate gene expression programs.

Training intensity-dependent and tissue-specific increases in lactate uptake and MCT-1 in heart and muscle.

We investigated the effects of 3 wk of moderate- (21 m/min, 8% grade) and highintensity treadmill training (31 m/min, 15% grade) on 1) monocarboxylate transporter 1 (MCT-1) content in rat hindlimb muscles and the heart and 2) lactate uptake in isolated soleus (Sol) muscles and perfused hearts. In the moderately trained group MCT-1 was not increased in any of the muscles [Sol, extensor digitorum longus (EDL), and red (RG) and white gastrocnemius (WG)] (P > 0.05). Similarly, lactate uptake in Sol strips was also not increased (P > 0.05). In contrast, in the heart, MCT-1 (+36%, P < 0.05) and lactate uptake (+72%, P < 0.05) were increased with moderate training. In the highly trained group, MCT-1 (+70%, P < 0.05) and lactate uptake (+79%, P < 0.05) were increased in Sol. MCT-1 was also increased in RG (+94%, P < 0.05) but not in WG and EDL (P > 0.05). In the highly trained group, heart MCT-1 (+44%, P < 0.05) and lactate uptake (+173%, P < 0.05) were increased. In conclusion, it has been shown that 1) in both heart and skeletal muscle lactate uptake is increased only when MCT-1 is increased; 2) training-induced increases in MCT-1 occurred at a lower training intensity in the heart than in skeletal muscle; 3) in the heart, lactate uptake was increased much more after high-intensity training than after moderate-intensity training, despite similar increases in heart MCT-1 with these two training intensities; and 4) the increases in MCT-1 occurred independently of any changes in the heart's oxidative capacity (as measured by citrate synthase activity).

Short-term training increases human muscle MCT1 and femoral venous lactate in relation to muscle lactate.

We examined the effects of increasing a known lactate transporter protein, monocarboxylate transporter 1 (MCT1), on lactate extrusion from human skeletal muscle during exercise. Before and after short-term bicycle ergometry training [2 h/day, 7 days at 65% maximal oxygen consumption (VO2max)], subjects (n = 7) completed a continuous bicycle ergometer ride at 30% VO2max (15 min), 60% VO2max (15 min), and 75% VO2max (15 min). Muscle biopsy samples (vastus lateralis) and arterial and femoral venous blood samples were obtained before exercise and at the end of each workload. After 7 days of training the MCT1 content in muscle was increased (+18%; P < 0.05). The concentrations of both muscle lactate and femoral venous lactate were reduced during exercise (P < 0.05) that was performed after training. High correlations were observed between muscle lactate and venous lactate before training (r = 0.92, P < 0.05) and after training (r = 0.85, P < 0.05), but the slopes of the regression lines between these variables differed markedly. Before training, the slope was 0.12 +/- 0.01 mM lactate.mmol lactate-1.kg muscle dry wt-1, and this was increased by 33% after training to 0.18 +/- 0.02 mM lactate.mmol lactate-1.kg muscle dry wt-1. This indicated that after training the femoral venous lactate concentrations were increased for a given amount of muscle lactate. These results suggest that lactate extrusion from exercising muscles is increased after training, and this may be associated with the increase in skeletal muscle MCT1.

Chronic electrical stimulation increases MCT1 and lactate uptake in red and white skeletal muscle.

We examined whether chronic stimulation of red and white rat muscles increased the concentrations of the monocarboxylate transporter MCT1. Red and white tibialis anterior (RTA and WTA, respectively) and extensor digitorum longus (EDL) muscles were chronically stimulated via the peroneal nerve for 7 days. Stimulated and contralateral control muscles were examined for MCT1 content, L-lactate uptake, lactate dehydrogenase (LDH) isoforms, and muscle fiber composition. MCT1 was 1.5 times greater in stimulated RTA, 3 times greater in stimulated WTA, and 1.9 times greater in stimulated EDL compared with respective control muscles (P < 0.05). L-Lactate uptake increased in all stimulated muscles (P < 0.05), and this was highly correlated with the increase in MCT1 (r = 0.96). The heart-type LDH (H-LDH) subunits also increased in all stimulated muscles (P < 0.05). The H-LDH subunits correlated highly with MCT1 in the muscles (r = 0.83). There was no change in muscle-type LDH subunits (P > 0.05). There were negligible alterations in muscle fiber composition in the stimulated muscles, suggesting that the increase in MCT1 was independent of changes in muscle fiber composition. These studies are the first to demonstrate that chronic muscle contraction increases MCT1 concentrations in both red and white skeletal muscles.

Cellular and subcellular expression of the monocarboxylate transporter MCT1 in rat heart. A high-resolution immunogold analysis.

An antibody to the C-terminus of the monocarboxylate transporter MCT1 was used to study the precise cellular and subcellular distribution of this transporter in rat heart. Postembedding immunogold procedures revealed that the labeling in the heart was restricted to cardiomyocytes and concentrated along the plasma membrane, including the transverse tubules. Gold particles occurred with highest densities in intercalated disks, where they avoided desmosomes and gap junctions. Labeling was also associated with plasmalemmal invaginations having ultrastructural features typical of caveolae. Internal membrane compartments were unlabeled. Quantitative analyses following postembedding labeling showed that the distribution of gold particles across the plasma membrane was nearly symmetrical, indicating that the C-terminus of the transporter is situated very close to the cell membrane. In preembedding immunogold experiments, the gold particles were localized at the external aspect of the plasma membrane, suggesting that the C-terminus is extracellular. From the present data, it can be concluded that even under basal conditions the majority of the MCT1 molecules in heart is present in the myocyte plasma membrane, implying that there is a constitutive functional expression of this transporter. It follows that the increased transmembrane flux of lactate during exercise or in pathological conditions such as ischemia must be a result of altered substrate gradients rather than of translocation of MCT1 molecules to the plasma membrane.

Effect of overexpressing GLUT-1 and GLUT-4 on insulin- and contraction-stimulated glucose transport in muscle.

To examine the effects of GLUT-1 on GLUT-4-dependent, insulin-stimulated, and contraction-stimulated 2-deoxy-D-glucose (2-DG) transport, we overexpressed GLUT-1 in metabolically heterogeneous skeletal muscles [red and white tibialis anterior (TA) and extensor digitorum longus (EDL)] via 7 days of chronic electrical stimulation. GLUT-1 was increased 1.6- to 16.4-fold (P < 0.05). Basal 2-DG transport was increased 1.7- to 3.0-fold (P < 0.05) and was equal to (red TA and EDL; P > 0.05) or exceeded insulin-stimulated 2-DG transport by 50% (white TA; P < 0.05) in the control muscles. GLUT-4 was concomitantly overexpressed (2.1- to 4.4-fold; P < 0.05). Insulin-stimulated 2-DG transport was increased 1.6- to 2.5-fold (P < 0.05). During muscle contractions, 2-DG transport increased 9- to 12-fold (P < 0.05) in control muscles, but this was reduced by approximately 25% (P < 0.05) in muscles overexpressing GLUT-1 and GLUT-4 (red TA and EDL). In contrast, in the experiment, white TA contraction-stimulated 2-DG transport was increased 1.7-fold (P < 0.05). Therefore, overexpression of GLUT-1, when GLUT-4 is also overexpressed, does not impair insulin-stimulated 2-DG transport, although contraction-stimulated transport may be reduced in some muscles.

Reciprocal GLUT-1 and GLUT-4 expression and glucose transport in denervated muscles.

We investigated in 3-day-denervated muscles 1) the expression of GLUT-1 in perineurial sheaths (PNS) and muscle, 2) the muscle fiber-specific changes in GLUT-1 and GLUT-4, and 3) changes in basal and insulin-stimulated 3-O-methylglucose transport. GLUT-1 was increased in both the PNS (P < 0.05) and in the muscle membranes (P < 0.05). GLUT-1 and GLUT-4 concentrations were changed reciprocally, in a fiber-dependent fashion [GLUT-1: red gastrocnemius (RG), +31%; white gastrocnemius (WG), +10%; GLUT-4: RG, -53%; WG, -16%]. Basal glucose transport was increased (P < 0.05), and this increase was correlated with the oxidative nature of the muscles (r = 0.97). Insulin-stimulated glucose transport was decreased in denervated muscles (P < 0.05). This was also related to the oxidative nature of the muscles (r = -0.88). The increase in basal glucose transport was correlated with the loss of insulin-stimulated transport (r = 0.95). Thus the increase in GLUT-1 compensates for the loss of GLUT-4, resulting in a 56% regain of the reduced insulin-stimulated glucose transport.

Role of the lactate transporter (MCT1) in skeletal muscles.

We used an antibody, constructed against the monocarboxylate transporter 1 (MCT1) protein (L. Carpenter, R. C. Poole, and A. P. Halestrap. Biochim. Biophys. Acta 1279: 157-165, 1996), to study the expression and role of MCT1 in rat skeletal muscles. MCT1 was higher in red than in white muscles (P < 0.05) and was highly correlated with the oxidative fiber content (%slow-twitch oxidative + %fast-twitch oxidative glycolytic) of skeletal muscles (r = 0.91). MCT1 was highly related to lactate uptake in skeletal muscles (r = 0.90). Total lactate dehydrogenase (LDH) activity, an index of glycolysis, was negatively correlated with MCT1 in rat muscles (r = -0.80). MCT1 was also strongly correlated with the heart-type forms of LDH (LDH-1 vs. MCT1, r = 0.83; LDH-2 vs. MCT1, r = 0.89). There was no relationship between MCT1 and the muscle form of LDH (LDH-5; P > 0.05). MCT1 was highly correlated with citrate synthase activity, a marker of the oxidative capacity of muscle (r = 0.82). Therefore, MCT1 may have kinetics that favor the uptake of L-lactate into the muscle cell for oxidative metabolism, and MCT1 may be coordinately expressed with the heart forms of LDH and enzymes of oxidative metabolism.

Chronic muscle stimulation increases lactate transport in rat skeletal muscle.

The aim of this study was to examine the effects of chronic low frequency stimulation on the lactate transport across the plasma membrane of the tibialis anterior (TA) muscle of the rat. Stimulating electrodes were implanted on either side of the peroneal nerve in one hindlimb. Chronic stimulation (10 Hz, 50 microsecond bursts, 24h/day) commenced 7 days after surgery, and were continued for 7 days. Animals were then left for 24 h, and thereafter muscles were obtained. Cytochrome C-oxidase activity was increased 1.9-fold in the stimulated TA compared to the control TA (p < 0.05). Lactate transport (zero-trans) was measured in giant sarcolemmal vesicles obtained from the chronically stimulated TA and the control TA. At each of the concentrations used in these studies a significant increase in lactate transport was observed; 2.8-fold increase at 1 mM lactate p < 0.05); 2-fold increases at both 30 mM and 50 mM lactate p < 0.05). These studies have shown that lactate transport capacity is markedly increased in response to chronic muscle contraction.

Reduced lactate transport in denervated rat skeletal muscle.

The purpose of this study was to investigate the effect of the neural regulation of contractile activity on lactate transport in skeletal muscle. Contractile activity of the rat soleus muscle was abolished by denervating the hindlimb muscles in one leg (3 days) while the sham-operated contralateral hindlimb muscles served as a control. Three days after surgery, lactate transport into the soleus muscle was measured in vitro, using incubated soleus muscle strips. Lactate uptake by the denervated soleus muscle was reduced compared with control (P < 0.05). The diffusive component of lactate transport was unaltered by denervation (P > 0.05). These results translated into a reduction in lactate carrier-mediated transport capacity (-68%) in the denervated soleus (P < 0.05). These studies indicate that loss of contractile activity results in a decrement of lactate transport, which is probably due to a reduction in the number of lactate carriers in the sarcolemma. Our results suggest that the inherent activity of the muscle is important in maintaining the lactate transport system.

L(+)-lactate binding to a protein in rat skeletal muscle plasma membranes.

Biochemical studies were conducted to determine the location of a putative lactate transport protein in rat skeletal muscle plasma membranes (PM). PM (50-100 micrograms protein) were incubated with [U-14C] L(+)-lactate, in the presence or absence of unlabeled monocarboxylates or potential inhibitors, after which proteins were separated by SDS-PAGE. Gel slices (2 mm) were cut and analyzed for 14C. [U-14C] L(+)-lactate was bound to plasma membranes in the 30 to 40 kDa molecular mass range. Binding of [U-14C] L(+)-lactate was inhibited by N-ethylmaleimide, unlabeled L-lactate and pyruvate, and in a dose dependent manner by alpha-cyano-4-hydroxycinnamate (r = 0.995), but not by cytochalasin-B. The inhibition of [U-14C] L(+)-lactate binding was similar to the inhibition of lactate transport. Therefore the transport of L(+)-lactate across skeletal muscle plasma membranes involves a polypeptide of 30 to 40 kDa.

Effects of exercise on lactate transport into mouse skeletal muscles.

Skeletal muscle lactate transport was investigated in vitro in isolated fast-twitch (EDL) and slow-twitch soleus (Sol) skeletal muscles from control and exercised mice. Exercise (23 m/min, 8% grade) reduced muscle glycogen by 37% in EDL (p < 0.05) and by 35% in Sol muscles (p < 0.05). Lactate transport measurements (45 sec) were performed after 60 min of exercise in intact EDL and Sol muscles in vitro, at differing pH (6.5 and 7.4) and differing lactate concentrations (4 and 30 mM). Lactate transport was observed to be greater in Sol than in EDL (p < 0.05). In the exercised muscles there was a small but significant increase in lactate transport (p < 0.05). Lactate transport was greater when exogenous lactate concentrations were greater (p < 0.05) and more rapid at the lower pH (p < 0.05). These studies demonstrated that lactate transport was increased with exercise.