Expression of neuronal NO synthase α- and ß-isoforms in skeletal muscle of mice.

Knowledge of the primary structure of neuronal NO synthase (nNOS) in skeletal muscle is still conflicting and needs further clarification. To elucidate the expression patterns of nNOS isoforms at both mRNA and protein level, systematic reverse transcription (RT)-PCR and epitope mapping by qualitative immunoblot analysis on skeletal muscle of C57/BL6 mice were performed. The ability of the nNOS isoforms to form aggregates was characterized by native low-temperature polyacrylamide electrophoresis (LT-PAGE). The molecular analysis was focused on the rectus femoris (RF) muscle, a skeletal muscle with a nearly balanced ratio of nNOS α- and ß-isoforms. RT-PCR amplificates from RF muscles showed exclusive exon-1d mRNA expression, either with or without exon-µ. Epitope mapping demonstrated the simultaneous expression of the nNOS splice variants α/µ, α/non-µ, ß/µ and ß/non-µ. Furthermore, immunoblotting suggests that the transition between nNOS α- and ß-isoforms lies within exon-3. In LT-PAGE, three protein nNOS associated aggregates were detected in homogenates of RF muscle and tibialis anterior muscle: a 320 kDa band containing nNOS α-isoforms, while 250 and 300 kDa bands consist of nNOS ß-isoforms that form homodimers or heterodimers with non-nNOS proteins.

Post-COVID exercise intolerance is associated with capillary alterations and immune dysregulations in skeletal muscles.

The SARS-CoV-2 pandemic not only resulted in millions of acute infections worldwide, but also in many cases of post-infectious syndromes, colloquially referred to as "long COVID". Due to the heterogeneous nature of symptoms and scarcity of available tissue samples, little is known about the underlying mechanisms. We present an in-depth analysis of skeletal muscle biopsies obtained from eleven patients suffering from enduring fatigue and post-exertional malaise after an infection with SARS-CoV-2. Compared to two independent historical control cohorts, patients with post-COVID exertion intolerance had fewer capillaries, thicker capillary basement membranes and increased numbers of CD169+ macrophages. SARS-CoV-2 RNA could not be detected in the muscle tissues. In addition, complement system related proteins were more abundant in the serum of patients with PCS, matching observations on the transcriptomic level in the muscle tissue. We hypothesize that the initial viral infection may have caused immune-mediated structural changes of the microvasculature, potentially explaining the exercise-dependent fatigue and muscle pain.

nNOS Increases Fiber Type-Specific Angiogenesis in Skeletal Muscle of Mice in Response to Endurance Exercise.

We studied the relationship between neuronal NO synthase (nNOS) expression and capillarity in the tibialis anterior (TA) muscle of mice subjected to treadmill training. The mRNA (+131%) and protein (+63%) levels of nNOS were higher (p ≤ 0.05) in the TA muscle of C57BL/6 mice undergoing treadmill training for 28 days than in those of littermates remaining sedentary, indicating an up-regulation of nNOS by endurance exercise. Both TA muscles of 16 C57BL/6 mice were subjected to gene electroporation with either the pIRES2-ZsGreen1 plasmid (control plasmid) or the pIRES2-ZsGreen1-nNOS gene-inserted plasmid (nNOS plasmid). Subsequently, one group of mice (n = 8) underwent treadmill training for seven days, while the second group of mice (n = 8) remained sedentary. At study end, 12-18% of TA muscle fibers expressed the fluorescent reporter gene ZsGreen1. Immunofluorescence for nNOS was 23% higher (p ≤ 0.05) in ZsGreen1-positive fibers than ZsGreen1-negative fibers from the nNOS-transfected TA muscle of mice subjected to treadmill training. Capillary contacts around myosin heavy-chain (MHC)-IIb immunoreactive fibers (14.2%; p ≤ 0.05) were only higher in ZsGreen1-positive fibers than ZsGreen1-negative fibers in the nNOS-plasmid-transfected TA muscles of trained mice. Our observations are in line with an angiogenic effect of quantitative increases in nNOS expression, specifically in type-IIb muscle fibers after treadmill training.

Promyelocytic leukemia protein promotes the phenotypic switch of smooth muscle cells in atherosclerotic plaques of human coronary arteries.

Promyelocytic leukemia protein (PML) is a constitutive component of PML nuclear bodies (PML-NBs), which function as stress-regulated SUMOylation factories. Since PML can also act as a regulator of the inflammatory and fibroproliferative responses characteristic of atherosclerosis, we investigated whether PML is implicated in this disease. Immunoblotting, ELISA and immunohistochemistry showed a stronger expression of PML in segments of human atherosclerotic coronary arteries and sections compared with non-atherosclerotic ones. In particular, PML was concentrated in PML-NBs from α-smooth muscle actin (α-SMA)-immunoreactive cells in plaque areas. To identify possible functional consequences of PML-accumulation in this cell type, differentiated human coronary artery smooth muscle cells (dHCASMCs) were transfected with a vector containing the intact PML-gene. These PML-transfected dHCASMCs showed higher levels of small ubiquitin-like modifier (SUMO)-1-dependent SUMOylated proteins, but lower levels of markers for smooth muscle cell (SMC) differentiation and revealed more proliferation and migration activities than dHCASMCs transfected with the vector lacking a specific gene insert or with the vector containing a mutated PML-gene coding for a PML-form without SUMOylation activity. When dHCASMCs were incubated with different cytokines, higher PML-levels were observed only after interferon γ (IFN-γ) stimulation, while the expression of differentiation markers was lower. However, these phenotypic changes were not observed in dHCASMCs treated with small interfering RNA (siRNA) suppressing PML-expression prior to IFN-γ stimulation. Taken together, our results imply that PML is a previously unknown functional factor in the molecular cascades associated with the pathogenesis of atherosclerosis and is positioned in vascular SMCs (VSMCs) between upstream IFN-γ activation and downstream SUMOylation.

Basement Membrane Remodeling Controls Endothelial Function in Idiopathic Pulmonary Arterial Hypertension.

The extracellular matrix (ECM) increasingly emerges as an active driver in several diseases, including idiopathic pulmonary arterial hypertension (IPAH). The basement membrane (BM) is a specialized class of ECM proteins. In pulmonary arteries, the BM is in close contact and direct proximity to vascular cells, including endothelial cells. So far, the role of the BM has remained underinvestigated in IPAH. Here, we aimed to shed light on the involvement of the BM in IPAH, by addressing its structure, composition, and function. On an ultrastructural level, we observed a marked increase in BM thickness in IPAH pulmonary vessels. BM composition was distinct in small and large vessels and altered in IPAH. Proteoglycans were mostly responsible for distinction between smaller and larger vessels, whereas BM collagens and laminins were more abundantly expressed in IPAH. Type IV collagen and laminin both strengthened endothelial barrier integrity. However, only type IV collagen concentration dependently increased cell adhesion of both donor and IPAH-derived pulmonary arterial endothelial cells (PAECs) and induced nuclear translocation of mechanosensitive transcriptional coactivator of the hippo pathway YAP (Yes-activated protein). On the other hand, laminin caused cytoplasmic retention of YAP in IPAH PAECs. Accordingly, silencing of COL4A5 and LAMC1, respectively, differentially affected tight junction formation and barrier integrity in both donor and IPAH PAECs. Collectively, our results highlight the importance of a well-maintained BM homeostasis. By linking changes in BM structure and composition to altered endothelial cell function, we here suggest an active involvement of the BM in IPAH pathogenesis.

Structural Microangiopathies in Skeletal Muscle Related to Systemic Vascular Pathologies in Humans.

It is unclear how microangiopathic changes in skeletal muscle vary among systemic vascular pathologies. We therefore analyzed the capillary fine structure in skeletal muscle from patients with arterial hypertension (HYPT), diabetes mellitus type 2 (T2DM) or intermittent claudication - peripheral arterial disease (IC/PAD). Tablet-based image analysis (TBIA) was carried out to largely re-evaluate 5,000 transmission electron micrographs of capillaries from 126 vastus lateralis biopsies of 75 individuals (HYPT, T2DM or IC/PAD patients as well as healthy individuals before and after endurance exercise training) used in previous morphometric studies, but assessed using stereological counting grids of different sizes. Serial block-face scanning electron microscopy (SBFSEM) of mouse skeletal muscle was used for validation of the particular fine structural events observed in human biopsies. The peri-capillary basement membrane (BM) was 38.5 and 45.5% thicker (P < 0.05) in T2DM and IC/PAD patients than in the other groups. A 17.7-39.6% lower (P < 0.05) index for intraluminal endothelial cell (EC) surface enlargement by projections was exclusively found in T2DM patients by TBIA morphometry. The proportion of capillaries with disrupted BM between pericytes (PC) and EC was higher (P < 0.05) in HYPT (33.2%) and T2DM (38.7%) patients than in the control group. Empty EC-sockets were more frequent (P < 0.05) in the three patient groups (20.6% in HYPT, 27.1% in T2DM, 30.0% in IC/PAD) than in the healthy individuals. SBFSEM confirmed that EC-sockets may exhibit close proximity to the capillary lumen. Our comparative morphometric analysis demonstrated that structural arrangement of skeletal muscle capillaries is more affected in T2DM than in HYPT or IC/PAD, although some similar elements of remodeling were found. The increased frequency of empty EC-sockets in the three patient groups indicates that the PC-EC interaction is commonly disturbed in these systemic vascular pathologies.

Capillary facilitation of skeletal muscle function in health and disease.

Skeletal muscle is highly vascularized, with perfusion being tightly regulated to meet wide-ranging metabolic demands. For decades, the capillary supply has been explored mainly in terms of evaluating the capillary numbers and their function in the supply of oxygen and substrates and the removal of metabolic byproducts. This review will focus on recent discoveries concerning the role played by capillaries in facilitating other aspects of cell regulation and maintenance, in health and disease, as well as alterations during the aging process. Novelty Capillaries play a central role in the coordination of the vascular response that controls blood flow during contraction and the cellular responses to which they feed into. Nitric oxide is an important regulatory compound within the cardiovascular system, and a significant contributor to skeletal muscle capillary angiogenesis and vasodilatory response to agonists. The microvascular network between muscle fibres may play a critical role in the distribution of signalling factors necessary for optimal muscle satellite cell function.

The effect of two exercise modalities on skeletal muscle capillary ultrastructure in individuals with type 2 diabetes.

Type 2 diabetes is associated with microvascular dysfunction, but little is known about how capillary ultrastructure is affected by exercise training. To investigate the effect of two types of exercise training on skeletal muscle capillary ultrastructure and capillarization in individuals with type 2 diabetes, 21 individuals with type 2 diabetes were allocated (randomized controlled trial) to 11 weeks of aerobic exercise training consisting of either moderate-intensity endurance training (END; n = 10) or low-volume high-intensity interval training (HIIT; n = 11). Skeletal muscle biopsies (m vastus lateralis) were obtained before and after the training intervention. At baseline, there was no difference in capillarization, capillary structure, and exercise hyperemia between the two groups. After the training intervention, capillary-to-fiber ratio increased by 8% ± 3% in the END group (P < 0.05) and was unchanged in the HIIT group with no difference between groups. Endothelium thickness increased (P < 0.05), basement membrane thickness decreased (P < 0.05), and the capillary lumen tended (P = 0.07) to increase in the END group, whereas these structural indicators were unchanged after HIIT. In contrast, skeletal muscle endothelial nitric oxide synthase (eNOS) increased after HIIT (P < 0.05), but not END, whereas there was no change in vascular endothelial growth factor (VEGF), superoxide dismutase (SOD)-2, or NADPH oxidase after both training protocols. In contrast to END training, HIIT did not alter capillarization or capillary structure in individuals with type 2 diabetes. In conclusion, HIIT appears to be a less effective strategy to treat capillary rarefaction and reduce basement thickening in type 2 diabetes.

Relation of nNOS isoforms to mitochondrial density and PGC-1alpha expression in striated muscles of mice.

The expression of neuronal NO synthase (nNOS) alpha- and beta-isoforms in skeletal muscle is well documented but only little information is available about their regulation/functions. Using different mouse models, we now assessed whether the expression of nNOS-isoforms in muscle fibers is related to mitochondria content/activity and regulated by peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha). Catalytic histochemistry revealed highest nNOS-concentrations to be present in type-2 oxidative muscle fibers. Differences in mitochondrial density between nNOS-KO-mice and WT-littermates established by morphometry after transmission electron microscopy were significant in the oxidative portion of the tibialis anterior muscle (TA) but not in rectus femoris muscle (RF) indicating an nNOS-dependent mitochondrial pool in TA. Quantitative immunoblotting displayed the nNOS alpha-isoform to preponderate in those striated muscles of C57BL/6-mice that comprise of many type-2 oxidative fibers, e.g. TA, while roughly even levels of the two nNOS-isoforms were expressed in those muscles that mainly consist of type-2 glycolytic fibers, e.g. RF. Differences in citrate synthase-activity in muscle homogenates between nNOS-KO-mice and WT-littermates were positively related to nNOS alpha-isoform levels. In transgenic-mice over-expressing muscular PGC-1alpha compared to WT-littermates, immunoblotting revealed a significant shift in nNOS-expression in favor of the alpha-isoform in six out of eight striated muscles (exceptions: soleus muscle and tongue) without consistent relationship to changes in the expression of mitochondrial markers. In summary, our study demonstrated the nNOS alpha-isoform expression to be related to mitochondrial content/activity and to be up-regulated by up-stream PGC-1alpha in striated muscles, particularly in those enriched with type-2 oxidative fibers implying a functional convergence of the two signaling systems in these fibers.

Increased capillary tortuosity and pericapillary basement membrane thinning in skeletal muscle of mice undergoing running wheel training.

To work out which microvascular remodeling processes occur in murine skeletal muscle during endurance exercise, we subjected C57BL/6 mice to voluntary running wheel training for 1 week (1 wk-t) or 6 weeks (6 wks-t). By means of morphometry, the capillarity as well as the compartmental and sub-compartmental structure of the capillaries were quantitatively described at the light microscopy level and at the electron microscopy level, respectively, in the plantaris (PLNT) muscle of the exercising mice in comparison to untrained littermates. In the early phase of the training (1 wk-t), angiogenesis [32% higher capillary/fiber (C/F) ratio; P<0.05] in PLNT muscle was accompanied by a tendency for capillary lumen enlargement (30%; P=0.06) and a reduction of the pericapillary basement membrane thickness [(CBMT) 12.7%; P=0.09] as well as a 21% shortening of intraluminal protrusion length (P<0.05), all compared with controls. After long-term training (6 wks-t), when the mice reached a steady state in running activity, additional angiogenesis (C/F ratio: 76%; P<0.05) and a 16.3% increase in capillary tortuosity (P<0.05) were established, accompanied by reversal of the lumen expansion (23%; P>0.05), further reduction of the CBMT (16.5%; P<0.05) and additional shortening of the intraluminal protrusion length (23%; P<0.05), all compared with controls. Other structural indicators, such as capillary profile sizes, profile area densities, perimeters of the capillary compartments and concentrations of endothelium-pericyte peg-socket junctions, were not significantly different between the mouse groups. Besides angiogenesis, increase of capillary tortuosity and reduction of CBMT represent the most striking microvascular remodeling processes in skeletal muscle of mice that undergo running wheel training.

Ultrastructure of Skeletal Muscles in Mice Lacking Muscle-Specific VEGF Expression.

Vascular endothelial growth factor-A (VEGF) influences several physiological processes including endothelial cell function, angiogenesis and maintenance of organ/tissue capillarity. While the functional aspects of VEGF were vigorously investigated, only little detail is known on structural integrity of skeletal muscle fibers and capillaries in mice lacking VEGF expression in their muscles. Therefore, we assessed systematically the architecture of the glycolytic plantaris and the oxidative soleus muscles obtained from muscle-specific VEGF knockout (mVEGF-KO, n = 7) mice and their wild-type (WT, n = 7) littermates by morphometry after transmission electron microscopy. The capillary/fiber ratio was lower (plantaris: -63.5%; soleus: -54.8%; P ≤ 0.05) in mVEGF-KO mice than in WT mice. In plantaris, quantification of volume density (Vv) of compartments revealed higher Vv of total mitochondria (+56.5%, P ≤ 0.05) as well as higher Vv-values for both intrafibrillar (+39%; P ≤ 0.05) and subsarcolemmal (+220%; P ≤ 0.05) mitochondrial pools in mVEGF-KO mice than WT mice. The capillary phenotype also differed (P ≤ 0.05) between the two mouse-strains: Vv (-17.4%), absolute area size (-19.1%) and thickness (-19.6%) of the endothelium layer were lower and Vv of capillary lumen (+15.1%) was higher in mVEGF-KO mice than in WT littermates. In soleus, mitochondrial Vv in fibers and the structural indicators specific to the capillary phenotype exhibited the same tendency in differences between the mouse strains without reaching statistical significance. Our morphometric analysis demonstrates that the lower capillary supply in plantaris of mVEGF-KO mice is accompanied by higher mitochondrial Vv in muscle fibers as well as lumen dilation and endothelium thinning of capillaries. These structural alterations were more pronounced in a glycolytic than an oxidative muscle. Anat Rec, 300:2239-2249, 2017. © 2017 Wiley Periodicals, Inc.

Pericapillary basement membrane thickening in human skeletal muscles.

The basement membrane (BM) surrounding capillaries in skeletal muscles varies physiologically in thickness according to age, physical fitness, and anatomical site in humans. Furthermore, the pericapillary BM thickness (CBMT) increases pathophysiologically during several common disease states, including peripheral arterial disease and diabetes mellitus. This review on CBM thickening in human skeletal muscles is two pronged. First, it addresses the advantages/disadvantages of grid- and tablet-based measuring and morphometric techniques that are implemented to assess the CBMT on transmission electron micrographs. Second, it deals with the biology of CBM thickening in skeletal muscles, particularly its possible causes, molecular mechanisms, and functional impact. CBM thickening is triggered by several physical factors, including diabetes-associated glycation, hydrostatic pressure, and inflammation. Increased biosynthesis of type IV collagen expression or repetitive cycles in pericyte or endothelial cell degeneration/proliferation appear to be most critical for CBM accumulation. A thickened CBM obviously poses a greater barrier for diffusion, lowers the microvascular elasticity, and impedes transcytosis of inflammatory cells. Our own morphometric data reveal the CBM enlargement to be not accompanied by the pericyte coverage. Owing to an overlap or redundancy in the capillary supply, CBM thickening in skeletal muscles might not be such a devastating occurrence as in organs with endarterial circulation (e.g., kidney and retina). CBM growth in skeletal muscles can be reversed by training or administration of antidiabetic drugs. In conclusion, CBM thickening in skeletal muscles is a microvascular remodeling process by which metabolic, hemodynamic, and inflammatory forces are integrated together and which could play a hitherto underestimated role in etiology/progression of human diseases.

Capillary ultrastructure and mitochondrial volume density in skeletal muscle in relation to reduced exercise capacity of patients with intermittent claudication.

Intermittent claudication (IC) is the most commonly reported symptom of peripheral arterial disease (PAD). Impaired limb blood flow is a major casual factor of lower exercise tolerance in PAD but cannot entirely explain it. We hypothesized that IC is associated with structural changes of the capillary-mitochondria interface that could contribute to the reduction of exercise tolerance in IC patients. Capillary and mitochondrial morphometry were performed after light and transmission electron microscopy using vastus lateralis muscle biopsies of 14 IC patients and 10 age-matched controls, and peak power output (PPO) was determined for all participants using an incremental single-leg knee-extension protocol. Capillary density was lower (411 ± 90 mm(-2) vs. 506 ± 95 mm(-2); P ≤ 0.05) in the biopsies of the IC patients than in those of the controls. The basement membrane (BM) around capillaries was thicker (543 ± 82 nm vs. 423 ± 97 nm; P ≤ 0.01) and the volume density of mitochondria was lower (3.51 ± 0.56% vs. 4.60 ± 0.74%; P ≤ 0.01) in the IC patients than the controls. In the IC patients, a higher proportion of capillaries appeared with collapsed slit-like lumen and/or swollen endothelium. PPO was lower (18.5 ± 9.9 W vs. 33.5 ± 9.4 W; P ≤ 0.01) in the IC patients than the controls. We suggest that several structural alterations in skeletal muscle, either collectively or separately, contribute to the reduction of exercise tolerance in IC patients.

Advances and challenges in skeletal muscle angiogenesis.

The role of capillaries is to serve as the interface for delivery of oxygen and removal of metabolites to/from tissues. During the past decade there has been a proliferation of studies that have advanced our understanding of angiogenesis, demonstrating that tissue capillary supply is under strict control during health but poorly controlled in disease, resulting in either excessive capillary growth (pathological angiogenesis) or losses in capillarity (rarefaction). Given that skeletal muscle comprises nearly 40% of body mass in humans, skeletal muscle capillary density has a significant impact on metabolism, endocrine function, and locomotion and is tightly regulated at many different levels. Skeletal muscle is also high adaptable and thus one of the few organ systems that can be experimentally manipulated (e.g., by exercise) to study physiological regulation of angiogenesis. This review will focus on the methodological concerns that have arisen in determining skeletal muscle capillarity and highlight the concepts that are reshaping our understanding of the angio-adaptation process. We also summarize selected new findings (physical influences, molecular changes, and ultrastructural rearrangement of capillaries) that identify areas of future research with the greatest potential to expand our understanding of how angiogenesis is normally regulated, and that may also help to better understand conditions of uncontrolled (pathological) angiogenesis.

Angiogenesis-related ultrastructural changes to capillaries in human skeletal muscle in response to endurance exercise.

The ultrastructure of capillaries in skeletal muscle was morphometrically assessed in vastus lateralis muscle (VL) biopsies taken before and after exercise from 22 participants of two training studies. In study 1 (8 wk of ergometer training), light microscopy revealed capillary-fiber (C/F) ratio (+27%) and capillary density (+16%) to be higher (P ≤ 0.05) in postexercise biopsies than in preexercise biopsies from all 10 participants. In study 2 (6 mo of moderate running), C/F ratio and capillary density were increased (+23% and +20%; respectively, P ≤ 0.05) in VL biopsies from 6 angiogenesis responders (AR) after training, whereas 6 nonangiogenesis responders (NR) showed nonsignificant changes in these structural indicators (-4%/-4%, respectively). Forty capillary profiles per participant were evaluated by point and intersection counting on cross sections after transmission electron microscopy. In study 1, volume density (Vv) and mean arithmetic thickness (T) of endothelial cells (ECs; +19%/+17%, respectively) and pericytes (PCs; +20%/+21%, respectively) were higher (P ≤ 0.05), whereas Vv and T of the pericapillary basement membrane (BM) were -23%/-22% lower (P ≤ 0.05), respectively, in posttraining biopsies. In study 2, exercise-related differences between AR and NR-groups were found for Vv and T of PCs (AR, +26%/+22%, respectively, both P ≤ 0.05; NR, +1%/-3%, respectively, both P > 0.05) and BM (AR, -14%/-13%, respectively, both P ≤ 0.05; NR, -9%/-11%, respectively, P = 0.07/0.10). Vv and T of ECs were higher (AR, +16%/+18%, respectively; NR, +6%/+6%, respectively; all P ≤ 0.05) in both groups. The PC coverage was higher (+13%, P ≤ 0.05) in VL biopsies of individuals in the AR group but nonsignificantly altered (+3%, P > 0.05) in those of the NR group after training. Our study suggests that intensified PC mobilization and BM thinning are related to exercise-induced angiogenesis in human skeletal muscle, whereas training per se induces EC-thickening.

Phenotype of capillaries in skeletal muscle of nNOS-knockout mice.

Because neuronal nitric oxide synthase (nNOS) has a well-known impact on arteriolar blood flow in skeletal muscle, we compared the ultrastructure and the hemodynamics of/in the ensuing capillaries in the extensor digitorum longus (EDL) muscle of male nNOS-knockout (KO) mice and wild-type (WT) littermates. The capillary-to-fiber (C/F) ratio (-9.1%) was lower (P ≤ 0.05) in the nNOS-KO mice than in the WT mice, whereas the mean cross-sectional fiber area (-7.8%) and the capillary density (-3.1%) varied only nonsignificantly (P > 0.05). Morphometrical estimation of the area occupied by the capillaries as well as the volume and surface densities of the subcellular compartments differed nonsignificantly (P > 0.05) between the two strains. Intravital microscopy revealed neither the capillary diameter (+3% in nNOS-KO mice vs. WT mice) nor the mean velocity of red blood cells in EDL muscle (+25% in nNOS-KO mice vs. WT mice) to significantly vary (P > 0.05) between the two strains. The calculated shear stress in the capillaries was likewise nonsignificantly different (3.8 ± 2.2 dyn/cm² in nNOS-KO mice and 2.1 ± 2.2 dyn/cm² in WT mice; P > 0.05). The mRNA levels of vascular endothelial growth factor (VEGF)-A were lower in the EDL muscle of nNOS-KO mice than in the WT littermates (-37%; P ≤ 0.05), whereas mRNA levels of VEGF receptor-2 (VEGFR-2) (-11%), hypoxia inducible factor-1α (+9%), fibroblast growth factor-2 (-14%), and thrombospondin-1 (-10%) differed nonsignificantly (P > 0.05). Our findings support the contention that VEGF-A mRNA expression and C/F-ratio but not the ultrastructure or the hemodynamics of/in capillaries in skeletal muscle at basal conditions depend on the expression of nNOS.

The impact of antioxidant supplements and endurance exercise on genes of the carbohydrate and lipid metabolism in skeletal muscle of mice.

To ascertain whether reactive oxygen species (ROS) contribute to training-induced adaptation of skeletal muscle, we administered ROS-scavenging antioxidants (AOX; 140 mg/l of ascorbic acid, 12 mg/l of coenzyme Q10 and 1% N-acetyl-cysteine) via drinking water to 16 C57BL/6 mice. Sixteen other mice received unadulterated tap water (CON). One cohort of both groups (CON(EXE) and AOX(EXE) ) was subjected to treadmill exercise for 4 weeks (16-26 m/min, incline of 5°-10°). The other two cohorts (CON(SED) and AOX(SED) ) remained sedentary. In skeletal muscles of the AOX(EXE) mice, GSSG and the expression levels of SOD-1 and PRDX-6 were significantly lower than those in the CON(EXE) mice after training, suggesting disturbance of ROS levels. The peak power related to the body weight and citrate synthase activity was not significantly influenced in mice receiving AOX. Supplementation with AOX significantly altered the mRNA levels of the exercise-sensitive genes HK-II, GLUT-4 and SREBF-1c and the regulator gene PGC-1alpha but not G6PDH, glycogenin, FABP-3, MCAD and CD36 in skeletal muscle. Although the administration of AOX during endurance exercise alters the expression of particular genes of the ROS metabolism, it does not influence peak power or generally shift the metabolism, but it modulates the expression of specific genes of the carbohydrate and lipid metabolism and PGC-1alpha within murine skeletal muscle.

The vascular-disrupting agent combretastatin impairs splitting and sprouting forms of physiological angiogenesis.

OBJECTIVE: Vascular-disrupting agents like combretastatin (CA-4-P), used to attenuate tumor blood flow in vivo, exert anti-mitotic and anti-migratory effects on endothelial cells in vitro. We tested whether anti-vascular or anti-angiogenic effects of CA-4-P are evident with physiological angiogenesis in skeletal muscle (EDL) due to sustained hyperemia (intraluminal splitting) and chronic muscle overload (abluminal sprouting). METHODS: CA-4-P was given i.v. (25 mg/kg on alternate days for 14 days) to mice subjected to angiogenic stimuli (prazosin or synergist extirpation). The responses of femoral artery blood flow as well as capillarity, capillary ultrastructure, and levels of Rho GTPase were measured. RESULTS: Blood flow was unaffected in the sprouting angiotype, but decreased in the splitting angiotype, by CA-4-P. In contrast, CA-4-P attenuated the capillarity increase in both models, associated with reduced lamellipodia and filopodia formation. Muscle overload, but not hyperemia, was accompanied by an increase in Rho GTPase with CA-4-P. CONCLUSIONS: CA-4-P impaired the angiogenic response in both experimental models. This inhibitory effect was associated with a lower increase in femoral blood flow in splitting, whereas sprouting angiogenesis was accompanied by higher Rho activity consistent with the interruption of actin polymerization. Thus, CA-4-P may exert context-dependent anti-vascular and anti-angiogenic effects in vivo under physiological conditions.

Exercise-induced angiogenesis correlates with the up-regulated expression of neuronal nitric oxide synthase (nNOS) in human skeletal muscle.

The contribution of neuronal nitric oxide synthase (nNOS) to angiogenesis in human skeletal muscle after endurance exercise is controversially discussed. We therefore ascertained whether the expression of nNOS is associated with the capillary density in biopsies of the vastus lateralis (VL) muscle that had been derived from 10 sedentary male subjects before and after moderate training (four 30-min weekly jogging sessions for 6 months, with a heart-rate corresponding to 75% VO(2)max). In these biopsies, nNOS was predominantly expressed as alpha-isoform with exon-mu and to a lesser extent without exon-mu, as determined by RT-PCR. The mRNA levels of nNOS were quantified by real-time PCR and related to the capillary-to-fibre ratio and the numerical density of capillaries specified by light microscopy. If the VL biopsies of all subjects were co-analysed, mRNA levels of nNOS were non-significantly elevated after training (+34%; P > 0.05). However, only five of the ten subjects exhibited significant (P ≤ 0.05) elevations in the capillary-to-fibre ratio (+25%) and the numerical density of capillaries (+21%) and were thus undergoing angiogenesis. If the VL biopsies of these five subjects alone were evaluated, the mRNA levels of nNOS were significantly up-regulated (+128%; P ≤ 0.05) and correlated positively (r = 0.8; P ≤ 0.01) to angiogenesis. Accordingly, nNOS protein expression in VL biopsies quantified by immunoblotting was significantly increased (+82%; P ≤ 0.05) only in those subjects that underwent angiogenesis. In conclusion, the expression of nNOS at mRNA and protein levels was statistically linked to capillarity after exercise suggesting that nNOS is involved in the angiogenic response to training in human skeletal muscle.

Remodeling of calcium handling in skeletal muscle through PGC-1α: impact on force, fatigability, and fiber type.

Regular endurance exercise remodels skeletal muscle, largely through the peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). PGC-1α promotes fiber type switching and resistance to fatigue. Intracellular calcium levels might play a role in both adaptive phenomena, yet a role for PGC-1α in the adaptation of calcium handling in skeletal muscle remains unknown. Using mice with transgenic overexpression of PGC-1α, we now investigated the effect of PGC-1α on calcium handling in skeletal muscle. We demonstrate that PGC-1α induces a quantitative reduction in calcium release from the sarcoplasmic reticulum by diminishing the expression of calcium-releasing molecules. Concomitantly, maximal muscle force is reduced in vivo and ex vivo. In addition, PGC-1α overexpression delays calcium clearance from the myoplasm by interfering with multiple mechanisms involved in calcium removal, leading to higher myoplasmic calcium levels following contraction. During prolonged muscle activity, the delayed calcium clearance might facilitate force production in mice overexpressing PGC-1α. Our results reveal a novel role of PGC-1α in altering the contractile properties of skeletal muscle by modulating calcium handling. Importantly, our findings indicate PGC-1α to be both down- as well as upstream of calcium signaling in this tissue. Overall, our findings suggest that in the adaptation to chronic exercise, PGC-1α reduces maximal force, increases resistance to fatigue, and drives fiber type switching partly through remodeling of calcium transients, in addition to promoting slow-type myofibrillar protein expression and adequate energy supply.