Skeletal Muscle Microvasculature Research Articles

Consideration of absolute intensity when examining sex differences in blood pressure responses during static exercise.

Low- to moderate-intensity submaximal static contractions are commonly used to study the effects of biological sex on the cardiovascular response to exercise. Under this paradigm, premenopausal females frequently demonstrate smaller blood pressure responses than age-matched males. These differences are preserved during postexercise circulatory occlusion, implicating the muscle metaboreflex as an important driver of sex differences in the blood pressure response to static exercise. The mechanisms responsible for these differences are incompletely understood but often attributed to innate sex differences in skeletal muscle fiber type distribution, muscle metabolism, and/or sympathetic control of the circulation. However, one potential confounding factor is that the majority of studies use relative intensity exercise (e.g., 30% of maximal voluntary contraction), such that on average, females are completing static contractions at a lower absolute intensity. In this review, we summarize human evidence showing that sex differences in blood pressure responses to static exercise are attenuated or abolished when controlling for absolute intensity and muscle strength, either by statistical methods or strength-matched cohorts. We highlight evidence that the effect of higher absolute contraction intensity on exercise blood pressure likely occurs through increased mechanical occlusion of skeletal muscle microvasculature, leading to greater activation of the muscle metaboreflex. These findings highlight an important need to account for absolute intensity when studying and interpreting sex differences in cardiovascular responses to exercise.

In vivo optical assessment of cerebral and skeletal muscle microvascular response to phenylephrine.

This study aimed to investigate the simultaneous response of the cerebral and skeletal muscle microvasculature to the same phenylephrine (PE) boluses. A hybrid optical system that combines hyperspectral near-infrared spectroscopy (hs-NIRS) and diffuse correlation spectroscopy (DCS) was used to monitor changes in tissue oxygenation and perfusion. Data were collected from the head and hind limb of seven male Sprague-Dawley rats while administering intravenous (IV) injections of PE or saline to all animals. The response to saline was used as a control. Skeletal muscle oxygenation decreased significantly after PE injection, while a statistically underpowered decrease in perfusion was observed, followed by an increase beyond baseline. Vascular conductance also decreased in the muscle reflecting the drug's vasoconstrictive effects. Tissue oxygenation and perfusion increased in the brain in response to PE. Initially, there was a sharp increase in cerebral perfusion but no changes in cerebral vascular conductance. Subsequently, cerebral flow and vascular conductance decreased significantly below baseline, likely reflecting autoregulatory mechanisms to manage the excess flow. Further, fitting an exponential function to the secondary decrease in cerebral perfusion and increase in muscular blood flow revealed a quicker kinetic response in the brain to adjust blood flow. In the skeletal muscle, PE caused a transient decrease in blood volume due to vasoconstriction, which resulted in an overall decrease in hemoglobin content and tissue oxygen saturation. Since PE does not directly affect cerebral vessels, this peripheral vasoconstriction shunted blood into the brain, resulting in an initial increase in oxygenated hemoglobin and oxygen saturation.

Mitochondrial and Microvascular Impairments in a Novel Swine Model of Peripheral Artery Disease

Introduction: Impaired skeletal muscle mitochondria and microvascular function have been reported in human peripheral artery disease (PAD). However, no large animal models of PAD have mimicked these impairments. Purpose: The purpose of this study was to examine the skeletal muscle microvasculature and mitochondria in a novel swine model of PAD. Hypothesis: We hypothesized that hindlimb ischemia leads to dysfunction of skeletal muscle microvessels and mitochondria. Methods: Ossabaw swine (n=5) with metabolic syndrome underwent endovascular coil occlusion of the right external iliac artery to generate hindlimb ischemia (ISC). The left leg served as a non-ischemic control (CON). After 4-weeks, biopsies were collected from both legs (lateral gastrocnemius). Skeletal muscle arterioles were assessed using video microscopy. Microvascular vasodilatory function was assessed in response to flow (Shear stress), acetylcholine (ACh), and sodium nitroprusside (SNP). Skeletal muscle mitochondrial function was assessed using high-resolution respirometry. Substrates were administered as follows: malate (M) & glutamate (G), adenosine diphosphate (ADP), succinate (S), oligomycin, and ascorbate (Asc) & N,N,N,N-tetramethyl-phenylenediamine (TMPD). Complex I state 2 (CI-S2) was assessed as M+G, complex I state 3 (CI-S3) was assessed as (M+G+ADP)-(M+G), complex II state 3 (CII-S3) was assessed as (M+G+ADP+S)-(M+G+ADP), complex I&II state 3 (CI&II S3) was assessed as (M+G+ADP+S)-(M+G), state 4 (S4) was assessed after oligomycin, and complex IV (CIV) respiration was assessed after Asc+TMPD. The respiratory control ratio (RCR) was calculated as CI&II-S3/S4. Results: Vasodilation in response to flow was reduced in ISC vs. CON (Δ-13.1±13.7%, p=0.01). but not in response to ACh (Δ-6.2±17.7%, p=0.23) or SNP (Δ1.7±11.0%, p=0.53). Additionally, CI-S2 (Δ-0.3±3.7 pmolO2·s−1·mg−1, p=0.65), S4 (Δ-1.7±4.4 pmolO2·s−1·mg−1, p=0.14), and CIV (Δ-2.8±20.9 pmolO2·s−1·mg−1, p=0.65) were not different between ISC and CON. However, CI-S3 (Δ-4.7±5.0 pmolO2·s−1·mg−1, p<0.01), CII-S3 (Δ-3.5±5.0 pmolO2·s−1·mg−1, p=0.02), CI&II-S3 (Δ-8.5±8.7 pmolO2·s−1·mg−1, p<0.01), and RCR (Δ-0.8±1.3 pmolO2·s−1·mg−1, p=0.04) were all reduced in ISC vs. CON. Conclusions: We found that skeletal muscle microvessels and mitochondria were impaired in our swine model. Based on the present findings, we conclude that our novel porcine model closely replicates the pathophysiology PAD, making it a promising large animal model for studying human PAD. This research was supported by the National Institutes of Health (R01HD106911, R01AG062198, R01AG0778031) and the Great Plains IdeA-CTR (U54 GM115458). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A constrained constructive optimization model of branching arteriolar networks in rat skeletal muscle.

Blood flow regulation within the microvasculature reflects a complex interaction of regulatory mechanisms and varies spatially and temporally according to conditions such as metabolism, growth, injury, and disease. Understanding the role of microvascular flow distributions across conditions is of interest to investigators spanning multiple disciplines; however, data collection within networks can be labor-intensive and challenging due to limited resolution. To overcome these experimental challenges, computational network models that can accurately simulate vascular behavior are highly beneficial. Constrained constructive optimization (CCO) is a commonly used algorithm for vascular simulation, particularly well known for its adaptability toward vascular modeling across tissues. The present work demonstrates an implementation of CCO aimed to simulate a branching arteriolar microvasculature in healthy skeletal muscle, validated against literature including comprehensive rat gluteus maximus vasculature datasets, and reviews a list of user-specified adjustable model parameters to understand how their variability affects the simulated networks. Network geometric properties, including mean element diameters, lengths, and numbers of bifurcations per order, Horton's law ratios, and fractal dimension, demonstrate good validation once model parameters are adjusted to experimental data. This model successfully demonstrates hemodynamic properties such as Murray's law and the network Fahraeus effect. Application of centrifugal and Strahler ordering schemes results in divergent descriptions of identical simulated networks. This work introduces a novel CCO-based model focused on generating branching skeletal muscle microvascular arteriolar networks based on adjustable model parameters, thus making it a valuable tool for investigations into skeletal muscle microvascular structure and tissue perfusion.NEW & NOTEWORTHY The present work introduces a CCO-based algorithm for generating branching arteriolar networks, with adjustable model parameters to enable modeling in varying skeletal muscle tissues. The geometric and hemodynamic parameters of the generated networks have been comprehensively validated using experimental data collected previously in-house and from literature. This is one of few validated CCO-based models to specialize in skeletal muscle microvasculature and acts as a beneficial tool for investigating the microvasculature for hypothesis testing and validation.

Assessment of power spectral density of microvascular hemodynamics in skeletal muscles at very low and low-frequency via near-infrared diffuse optical spectroscopies.

In this work, we used a hybrid time domain near-infrared spectroscopy (TD-NIRS) and diffuse correlation spectroscopy (DCS) device to retrieve hemoglobin and blood flow oscillations of skeletal muscle microvasculature. We focused on very low (VLF) and low-frequency (LF) oscillations (i.e., frequency lower than 0.145 Hz), that are related to myogenic, neurogenic and endothelial activities. We measured power spectral density (PSD) of blood flow and hemoglobin concentration in four muscles (thenar eminence, plantar fascia, sternocleidomastoid and forearm) of 14 healthy volunteers to highlight possible differences in microvascular hemodynamic oscillations. We observed larger PSDs for blood flow compared to hemoglobin concentration, in particular in case of distal muscles (i.e., thenar eminence and plantar fascia). Finally, we compared the PSDs measured on the thenar eminence of healthy subjects with the ones measured on a septic patient in the intensive care unit: lower power in the endothelial-dependent frequency band, and larger power in the myogenic ones were observed in the septic patient, in accordance with previous works based on laser doppler flowmetry.

Skeletal muscle microcirculatory and mitochondrial function in patients with claudication and critical limb ischemia

Introduction: Peripheral artery disease (PAD) is an atherosclerotic progressive disorder that impairs blood flow to the lower extremities. Early-stage patients often present with intermittent claudication (leg pain during walking, CLD), but as the disease progresses, patients can experience pain at rest and tissue death (critical limb ischemia, CLI). We recently reported that skeletal muscle microvascular endothelial dysfunction presented in claudicating patients with PAD. However, it is unknown if this microvascular dysfunction continues to progress with disease severity. Objective: To compare a direct investigation of the skeletal muscle microcirculation and the mitochondrial respiratory environment in patients with claudication and patients with CLI. Hypothesis: We hypothesize that (1) CLI will have greater attenuation in microcirculatory endothelial-dependent and endothelial-independent vasodilatory function (smooth muscle function) than CLD and (2) impairments in skeletal muscle microcirculatory vasodilatory function will contribute to attenuated mitochondrial respiratory function in CLD and CLI. Methods: Medial gastrocnemius muscles were harvested from PAD patients with CLD ( n = 5), CLI ( n = 6), and age-matched controls ( n = 6) by both muscle biopsies and open incisions. Arterioles were isolated to assess endothelium-dependent and independent vasodilatory responses to flow (30 mL·min -1 ), acetylcholine (ACh), and sodium nitroprusside (SNP). Skeletal muscle mitochondrial respiratory function was determined by high-resolution respirometry. Results: Endothelium-dependent vasodilation was attenuated ( P < 0.05) in response to flow (CON: 53.6 ± 23%, PAD: 26.7 ± 10.4%, CLI: 20.1 ± 14.5%) and ACh (10 -3 M, CON: 64 ± 6.9%, PAD: 39.1 ± 6.7%, CLI: 20.1 ± 14.5%) in both PAD and CLI compared to CON. However, endothelium-independent vasodilation with SNP was not different in any condition ( P > 0.05). Complex I+II state 3 mitochondrial respiration was blunted in both CLD and CLI compared to CON ( P < 0.05). However, state 4 respiration in CON was greater than CLI ( P < 0.05) but lower than CLD. Additionally, complex I+II state 3 respiration was positively associated with both flow ( r = 0.5, P = 0.01) and ACh mediated vasodilation ( r = 0.6, P = 0.009). No differences were found between CLD and CLI conditions. Conclusions: This was the first the first study to directly examine and compare skeletal muscle microvascular function in PAD patients with CLD and CLI. Our initial data indicate that functional characteristics of the skeletal muscle microvasculature and mitochondria are altered in PAD and deteriorate with disease progression. There is a correlation between alterations in the endothelium-dependent microvessel function and the dysfunction of mitochondria in the ischemic muscle raising the possibility that the microvascular dysfunction in PAD is produced by defects in the mitochondria of the microvascular endothelium. National Institutes of Health (R01 AG034995, R01 AG049868) NIH National Institute of General Medical Sciences, which funds the Great Plains IdeA-CTR Network (U54 GM115458) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mesoangioblasts at 20: From the embryonic aorta to the patient bed.

In 2002 we published an article describing a population of vessel-associated progenitors that we termed mesoangioblasts (MABs). During the past decade evidence had accumulated that during muscle development and regeneration things may be more complex than a simple sequence of binary choices (e.g., dorsal vs. ventral somite). LacZ expressing fibroblasts could fuse with unlabelled myoblasts but not among themselves or with other cell types. Bone marrow derived, circulating progenitors were able to participate in muscle regeneration, though in very small percentage. Searching for the embryonic origin of these progenitors, we identified them as originating at least in part from the embryonic aorta and, at later stages, from the microvasculature of skeletal muscle. While continuing to investigate origin and fate of MABs, the fact that they could be expanded in vitro (also from human muscle) and cross the vessel wall, suggested a protocol for the cell therapy of muscular dystrophies. We tested this protocol in mice and dogs before proceeding to the first clinical trial on Duchenne Muscular Dystrophy patients that showed safety but minimal efficacy. In the last years, we have worked to overcome the problem of low engraftment and tried to understand their role as auxiliary myogenic progenitors during development and regeneration.

Angiostatic freeze or angiogenic move? Acute cold stress prevents angiokine secretion from murine myotubes but primes primary endothelial cells for greater migratory capacity.

The skeletal muscle tissue can adapt to exercise and environmental stressors with a remarkable plasticity. Prolonged cold stress exposure has been associated to increased skeletal muscle capillarization. Angioadaptation refers to the coordinated molecular and cellular processes that influence the remodeling of skeletal muscle microvasculature. Two cell types are central to angioadaptation: the myocytes, representing an important source of angiokines; and the skeletal muscle endothelial cell (SMECs), targets of these angiokines and main constituents of muscle capillaries. The influence of cold stress on skeletal muscle angioadaptation remains largely unknown, particularly with respect to myocyte-specific angiokines secretion or endothelial cell angioadaptive responses. Here, we use an in vitro model to investigate the impact of cold stress (28°C versus 37°C) on C2C12 myotubes and SMECs. Our main objectives were to evaluate: 1) the direct impact of cold stress on C2C12 cellular expression of angiokines and their release in the extracellular environment; 2) the indirect impact of cold stress on SMECs migration via these C2C12-derived angiokines; and 3) the direct effect of cold stress on SMECs angioadaptive responses, including migration, proliferation, and the activation of the vascular endothelial growth factor receptor-2 (VEGFR2). Cold stress reduced the secretion of angiokines in C2C12 myotubes culture media irrespective their pro-angiogenic or angiostatic nature. In SMECs, cold stress abrogated cell proliferation and reduced the activation of VEGFR2 despite a greater expression of this receptor. Finally, SMECs pre-conditioned to cold stress displayed an enhanced migratory response when migration was stimulated in rewarming conditions. Altogether our results suggest that cold stress may be overall angiostatic. However, cold stress accompanied by rewarming may be seen as a pro-angiogenic stressor for SMECs. This observation questions the potential for using pre-cooling in sport-performance or therapeutic exercise prescription to enhance skeletal muscle angioadaptive responses to exercise.

P-034: EFFECTS OF HYDROXYUREA ON SKELETAL MUSCLE MICROVASCULATURE OF MICE WITH SICKLE CELL DISEASE

P-034: EFFECTS OF HYDROXYUREA ON SKELETAL MUSCLE MICROVASCULATURE OF MICE WITH SICKLE CELL DISEASE

Quantifying Longitudinal Network Geometry Alterations in Rat Skeletal Microvasculature Subject to Ischemia

Study objectiveIntra‐vital video microscopy (IVVM) data of rat subcutaneous skeletal muscle microvasculature under ischemic conditions at two time points (0 hours, 3 hours) was obtained from our collaborator. The objectives of this study are to elicit network geometries from the IVVM data and quantify the alterations in the network statistics of the geometries due to ischemia.HypothesisWe hypothesize that ischemia causes blood vessel de‐recruitment.MethodsIVVM data from six experiments was obtained, where each experiment provided a video at the start (0 hours) and end (3 hours) of the experiment. Using consecutive still images from the videos, vessel centerlines were manually drawn using Procreate and the NIH software, ImageJ. The included vessels were chosen based on two features: (1) visible red blood cell flow and (2) clearly defined edges. The centerlines were used to guide thresholding by identifying appropriate vessels. This was followed by the generation of binarized representations of the microvascular networks. The network statistics were quantified by measuring vessel segment lengths and diameters, counting the number of bifurcations, and calculating vessel density in ImageJ.ResultsAt 3 hours, the average vessel length changed from 283.91 + 252.14 pixels to 316.75 + 292.37 pixels, the average diameter changed from 16.02 + 7.99 pixels to 15.42 + 7.50 pixels, the average number of bifurcations changed from 32.42 to 18.92 and the average vessel density changed from 0.0085 + 0.0017 to 0.0061 + 0.0024.ConclusionThere is a clear decrease in average diameter, vessel density and the number of bifurcations characterizing the microvascular networks. However, an increase in the average vessel length accompanies these findings. The data indicates that ischemia may cause de‐recruitment of blood vessels.

Glial cell line‐derived neurotrophic factor and basic fibroblast growth factor derived from skeletal muscle pericytes increase the barrier function of endothelial cells in the endomysium

AbstractObjectivesIn skeletal muscle, the capillaries have tight junctions (TJs) that are structurally similar to those in the blood‐brain barrier (BBB) and blood‐nerve barrier (BNB). Although the endomysial capillaries include endothelial cells and pericytes, the properties of the endomysial pericytes have not been well elucidated. The purpose of this study was to establish a human skeletal muscle pericyte cell (HSMPCT) line, and examine whether the established cell line has similar properties to pericytes from the BBB and BNB.MethodHSMPCTs were isolated and introduced with retroviruses harboring temperature‐sensitive SV40 T antigen and telomerase genes. Then, we examined whether this cell line modifies the barrier function of a human skeletal muscle microvascular endothelial cell line, TSM15.ResultsA new HSMPCT cell line was successfully established. The conditioned media (CM) from HSMPCTs increased the transendothelial electrical resistance of the TSM15 cells and decreased permeability to 10kD‐dextran. Glial cell line‐derived neurotrophic factor (GDNF) and basic fibroblast growth factor (bFGF) increased the barrier function of TSM15 cells. The CM of HSMPCTs with anti‐GDNF or anti‐bFGF antibodies attenuated the effect of upregulating the barrier function of TSM15 cells. These results indicate that soluble factors, such as GDNF and bFGF, released from HSMPCTs strengthen the barrier function of TSM15 cells.ConclusionGDNF and bFGF released from HSMPCTs increase the barrier function of endomysial endothelium. These HSMPCTs and TSM15 cells, which are recognized as an in vitro model of the endomysial capillary, might facilitate the analysis of the pathophysiology of the microvasculature in human skeletal muscle.

Regenerated Microvascular Networks in Ischemic Skeletal Muscle.

Skeletal muscle is the largest organ in humans. The viability and performance of this metabolically demanding organ are exquisitely dependent on the integrity of its microcirculation. The architectural and functional attributes of the skeletal muscle microvasculature are acquired during embryonic and early postnatal development. However, peripheral vascular disease in the adult can damage the distal microvasculature, together with damaging the skeletal myofibers. Importantly, adult skeletal muscle has the capacity to regenerate. Understanding the extent to which the microvascular network also reforms, and acquires structural and functional competence, will thus be critical to regenerative medicine efforts for those with peripheral artery disease (PAD). Herein, we discuss recent advances in studying the regenerating microvasculature in the mouse hindlimb following severe ischemic injury. We highlight new insights arising from real-time imaging of the microcirculation. This includes identifying otherwise hidden flaws in both network microarchitecture and function, deficiencies that could underlie the progressive nature of PAD and its refractoriness to therapy. Recognizing and overcoming these vulnerabilities in regenerative angiogenesis will be important for advancing treatment options for PAD.

The Capillary‐Pericyte Relationship in Skeletal Muscle: Implications for Blood Flow Control

Introduction Pericytes, mural cells of the vasculature, are found adjacent to endothelial cells of capillaries and venules. In the brain, pericytes have been shown to be involved in blood flow control to active nerve tissue. In skeletal muscle, capillaries have been found to be important in directing blood flow to active skeletal muscle fibres. Active skeletal muscle fibres signal capillary endothelial cells to initiate signals that are transmitted upstream, along the blood vessel wall, to cause vasodilation of the upstream arteriole controlling the perfusion of the stimulated capillary. Pericytes have been shown to be associated with the microvasculature in skeletal muscle. If pericytes are closely associated with the capillary wall and capillary endothelial cells, they may be involved in the transmission of the upstream arteriolar vasodilatory signals that control capillary perfusion and blood flow to active skeletal muscle fibres. In order for this to be possible pericytes would have to have to form a continuous, connected layer of cells adjacent to the capillary network such that signals could be passed from pericyte to pericyte upstream to associated arterioles. In order to investigate the potential role of pericytes in blood flow control to skeletal muscle capillaries we investigated 1) whether pericytes were associated with capillaries in skeletal muscle and 2) whether pericytes form a continuous layer adjacent to the capillary network. Methods We used histological techniques, sectioning male mouse slow-oxidative (soleus (SOL)), and fast-oxidative-glycolytic muscle (diaphragm (DIA)) both cross sectionally and longitudinally in order to identify capillary endothelial cells (using isolectin) in cross section and to view capillaries along their length. We then double stained these sections with neural-glial antigen 2 (NG2) to identify the association of pericytes with capillary endothelial cells. Results Cross sectional analysis of 3 SOL muscles (273 capillaries counted) showed that 63.2+/-4.9% (avg+/- std. dev.) of capillaries were associated with pericytes while 36.8+/-4.9% of capillaries were not associated with pericytes. Cross sectional analysis of 2 DIA muscles (208 capillaries counted) showed that 53.6+/-7.7% of capillaries were associated with pericytes while 46.4+/-7.7% of capillaries were not associated with pericytes. In both muscle types, analysis of longitudinal sections, where capillaries could be viewed along their length, showed a lack of consistent association with pericytes. Conclusions We identified that, while there was an association between pericytes and capillary endothelial cells in skeletal muscle, not all capillary endothelial cells were associated with pericytes. Further, when capillaries were viewed along their length, pericytes were not consistently located adjacent to the capillary network. Our data indicate that pericytes may not form a continuous layer alongside the capillary network and may not be able to conduct vasodilatory signals from pericyte to pericyte to communicate with upstream arterioles and, therefore, may not be able to alter blood flow to active skeletal muscle fibres.

Preliminary Data Monitoring In Vivo Sources of Reactive Oxygen Species Production in Human Obesity

Introduction Excessive reactive oxygen species (ROS) production is a prominent feature of obesity-related conditions including impairments in endothelial function. NADPH oxidases (Nox) and mitochondria are major sources of ROS production within the vasculature. However, studies involving direct measurement of in vivo ROS production are limited in humans. Thus, the relative contributions of Nox- and mitochondrial-derived ROS in human obesity remain to be fully characterized. The objective of this study was to determine the predominant sources of in vivo ROS production in the skeletal muscle microvasculature of individuals with obesity. We hypothesize that both Nox- and mitochondria-dependent ROS production are upregulated in human obesity. Methods Two sedentary men with Class II and above obesity (body mass index ≥ 35 kg/m2) and the Metabolic Syndrome were studied. Microdialysis was used for measurements of in vivo skeletal muscle ROS production. Microdialysis probes were perfused with saline containing Amplex Ultrared, horseradish peroxidase and superoxide dismutase to measure local hydrogen peroxide and superoxide concentrations. To assess ROS production from distinct sources, microdialysis probes were perfused with the following: apocynin (non-specific Nox inhibitor), MitoTEMPO (mitochondrial-ROS inhibitor), and GKT 137831 (specific Nox 4 inhibitor). Results The concentrations of hydrogen peroxide and superoxide decreased upon addition of apocynin to microdialysis probes (mean ± SEM; hydrogen peroxide: 1.18 ± 0.58 to 0.76 ± 0.01 μM; superoxide: 1.47 ± 0.46 to 1.10 ± 0.62 μM). MitoTEMPO perfusion elicited an increase in hydrogen peroxide (1.12 ± 0.76 to 1.51 ± 1.08 μM) and decrease in superoxide (1.47 ± 0.46 to 0.78 ± 0.10 μM). The combination of apocynin plus MitoTEMPO resulted in concentrations of hydrogen peroxide (0.22 ± 0.10 μM) and superoxide (0.23 ± 0.06 μM) that were 69.7% and 79.1% lower than apocynin alone. In addition, the hydrogen peroxide (0.56 ± 0.25 μM) and superoxide (0.43 ± 0.18 μM) concentrations with co-perfusion of apocynin and GKT were 26.3% and 60.9% lower than with apocynin alone. Conclusions Our preliminary data suggest that mitochondria and Nox together are significant sources of ROS production within the skeletal muscle microvasculature of obese individuals. The local inhibition of Nox- and mitochondrial-derived ROS resulted in marked decreases in extracellular ROS levels within the skeletal muscle microvasculature. Although the small sample size currently limits our interpretation of the results, this study is anticipated to become the first to report on distinct sources of in vivo ROS production in human obesity.

Evaluation Of Functional Sympatholysis Occurring Within Contracting Skeletal Muscle Microvasculature In Humans

The ability of contracting skeletal muscle to blunt sympathetic vasoconstriction, termed functional sympatholysis, is thought to contribute to supplying adequate blood to the exercising muscle despite increased sympathetic tone. However, previous studies in humans primarily investigated vascular responses of exercising limb as a whole that reflects not only responses of contracting skeletal muscles but also responses of inactive tissues such as skin, adipose and bones. PURPOSE: We aimed to more directly investigate the functional sympatholysis occurring within exercising skeletal muscle in humans. METHODS: In 26 healthy male volunteers, we examined the effects of acute sympathoexcitation by 90-s forehead cooling at rest and during dynamic handgrip exercise at 10% and 30% of maximum voluntary contraction (10%Ex and 30%Ex), respectively. The handgrip exercises were continued 3 min and forehead cooling was applied at latter half of the exercise period. The subjects also performed handgrip exercises without forehead cooling as control conditions. We employed near-infrared diffuse correlation spectroscopy, an emerging optical technique for noninvasive measurement of deep tissue hemodynamics, to continuously measure the microcirculatory blood flow index (BFI) within the flexor digitorum superficialis muscle, the muscle primarily responsible for handgrip. RESULTS: The acute sympathoexcitation induced significant decrease in vascular conductance (BFI / mean arterial pressure) at rest (baseline vs. forehead cooling: 1.00 ± 0 vs. 0.75 ± 0.03 AU, p < 0.05) and during 10%Ex (control vs. forehead cooling: 1.66 ± 0.08 vs. 1.32 ± 0.10 AU, p < 0.05), but not during 30%Ex (control vs. forehead cooling: 4.00 ± 0.56 vs. 3.66 ± 0.50 AU, p > 0.05). In addition, the percentage reduction in vascular conductance by forehead cooling was significantly decreased during 30%Ex compared to rest (-25.2 ± 2.5 vs. -4.9 ± 5.1 %, p < 0.05). CONCLUSIONS: Our study demonstrated the functional sympatholysis occurring within the contracting skeletal muscle microvasculature in humans. Furthermore, our results revealed the clear intensity-dependent response such that mild exercise hardly interferes with sympathetic vasoconstriction, whereas moderate exercise substantially attenuates it.

228-LB: Chronic Treatment with a Vasodilator Augments the Effectiveness of Endurance Training to Improve Exercise Capacity and Increases Microvascular Blood Flow in Obese Mice

Peripheral vascular disease is a frequent co-morbidity of type-2 diabetes that impairs exercise capacity. We hypothesized that chronic treatment with a vasodilator would increase microvascular flow and exercise capacity. To test this hypothesis, we chronically treated obese mice with the vasodilatory agent Sildenafil during exercise training. Obese mice were fed a high fat diet (HFD) for 6 weeks prior to the study and remained on the diet for its duration. Mice performed an initial treadmill stress test to assess exercise capacity and injected twice daily with either vehicle or Sildenafil. Groups were matched to initial stress test run distance (Sildenafil 379±74 m vs. Vehicle 373±65 m n=17). Mice were exercise trained for 5-weeks with a 5-days per week 1-hour run at 40% of maximal stress test exercise intensity. After the training period, mice were subjected to a second exercise stress test. Sildenafil treated but not vehicle treated HFD-fed mice showed increased exercise capacity based on stress test run distance (Sildenafil 520±130 m vs. Vehicle 402±164 m n=17). Treating obese mice with a single acute dose of Sildenafil did not alter run distance on a treadmill stress test (Sildenafil 406±64 m vs. Vehicle 460±136 m n=8). To determine the effect of chronic Sildenafil treatment on vascular function, we measured blood flow in the microvasculature of the gastrocnemius muscle by intravital microscopy within one week of the final exercise test. Sildenafil treated mice showed increased vascular flow rate (Sildenafil 671±106 microns/s vs. Vehicle 527±198 microns/s, n=10-11) and vessel perfusion (Sildenafil 96±4% vs. Vehicle 92±4%, n=10-11). Overall, these results indicate that chronic treatment with a vasodilatory agent augments the effect of exercise training to improve exercise capacity in the context of obesity and increases blood flow rate and blood vessel perfusion in the microvasculature of skeletal muscle. Disclosure D.A. Cappel: None. D. Wasserman: None. Funding National Institutes of Health (DK059637, DK054902); American Heart Association

972-P: Liraglutide Attenuates Lipid-Induced Microvascular Insulin Resistance in Humans

Insulin recruits skeletal and cardiac muscle microvasculature to increase insulin and nutrient delivery, but this action is lost in insulin resistant states. Glucagon-like peptide 1 (GLP-1) improves microvascular perfusion in humans with obesity and the GLP-1 receptor agonist liraglutide attenuates muscle microvascular insulin resistance (IR) in rodents. To examine whether GLP-1 receptor agonism improves insulin’s microvascular actions in humans with acute IR, 18 healthy human subjects randomly received a systemic infusion of either saline or lipid emulsion (20%, 45 mL x 1 h and then 30 mL x 4 h) for 5 h or lipid emulsion for 5 h + liraglutide pre-treatment (0.6, 1.2, then 1.8 mg daily SC each x 2 days) with euglycemic insulin clamp (1 mU/kg/min) superimposed for the last 120 min. Skeletal and cardiac muscle microvascular blood volume (MBV), flow-mediated dilation (FMD), pulse wave velocity (PWV), and augmentation index (AI) were determined before and after insulin clamp. Compared with saline admission, insulin failed to increase MBV following lipid infusion in both skeletal (28.4 ± 14.1% vs. -1.3 ± 9.6% respectively, p=0.011) and cardiac (28.4 ± 14.1% vs. -1.3 ± 8.9%, p=0.027) muscle, consistent with acute IR induced by lipid infusion in both microvascular beds. Liraglutide pre-treatment improved insulin-mediated increases in MBV in the presence of lipid infusion in skeletal (8.9 ± 13.5%, p=0.117 vs. saline) and cardiac (12.6 ± 12.1%, p=0.101 vs. saline) muscle. There was no significant change in PWV, AI, or FMD amongst admissions. Glucose infusion rates were not significantly different amongst admissions (saline 6.02 ± 0.52, lipid 5.58 ± 0.62, vs. liraglutide + lipid 5.46 ± 0.46 mg/kg/min). In conclusion, liraglutide pretreatment effectively ameliorates lipid-induced skeletal and cardiac muscle microvascular IR in healthy humans. These data provide a mechanistic insight into the metabolic and cardiovascular benefits of GLP-1 receptor agonism seen in humans with obesity and type 2 diabetes. Disclosure K. Love: None. L. Jahn: None. L. Hartline: None. Z. Liu: None. Funding American Diabetes Association (1-17-ICST-059 to Z.L.); National Institutes of Health (1R01DK102359-01A1)

Effects of neuropeptide Y on the microvasculature of human skeletal muscle

BackgroundNeuropeptide Y acts directly on the vasculature as a cotransmitter with norepinephrine for an augmented contraction. Little, however, is known about the effects of neuropeptide Y on the microvasculature of human skeletal muscle. Neuropeptide Y signaling has not been studied in the setting of cardiac surgery and cardiopulmonary bypass. We investigated the role of neuropeptide Y signaling on vasomotor tone in the microvessels of human skeletal muscle, as well as the effect of cardiopulmonary bypass on neuropeptide Y-induced responsiveness. MethodsSpecimens taken from intercostal muscles were collected from patients, pre- and post-cardiopulmonary bypass, undergoing coronary artery bypass grafting or cardiac valve surgery (n = 8/group). Microvessels (157 ± 47 microns) were isolated in vitro in a no-flow state. Arterial microvascular responses to a neuropeptide Y agonist, a Y1 receptor antagonist, phenylephrine, and the coadministration of neuropeptide Y and phenylephrine were examined. The abundance and localization of the Y1 receptor were measured using Western blot and immunofluorescence, respectively. ResultsArterial microvessels showed responsiveness to the neuropeptide Y agonist (10–9 to 4 × 10–7 mol/L) both before and after cardiopulmonary bypass, reaching a 12.5% vasoconstriction from the baseline luminal diameter. With administration of the Y1 receptor antagonist after neuropeptide Y, the contractile response was eliminated (n = 3/group, P = .04). No difference in vasoconstriction was observed between pre- and post-cardiopulmonary bypass groups (P = .73). The coadministration of neuropeptide Y and phenylephrine (10–9 to 10–4 mol/L) elicited no difference in vasoconstriction (n = 7/group, P = .06 both pre- and post-cardiopulmonary bypass) when compared with phenylephrine alone (10–9 to 10–4 mol/L). No change in the protein expression or localization of the Y1 receptor was detected by Western blotting (n = 6/group, P = .44) or immunofluorescence (n = 6/group, P = .13). ConclusionNeuropeptide Y induced vasoconstriction, suggesting that neuropeptide Y may play an important role in the regulation of the peripheral microvasculature. There was no change in microvascular responsiveness to neuropeptide Y after cardiopulmonary bypass nor were there any synergistic effects of neuropeptide Y on phenylephrine-induced vasoconstriction in the skeletal muscle microvasculature.

Ginkgo biloba extract ameliorates aging-related sarcopenia

Background & Aim Sarcopenia, a geriatric syndrome named by Irwin Rosenberg in 1989, characterized by loss of muscle mass and muscle strength as well as by declination of muscle repair and regeneration. Sarcopenia not only contributes to the progressive loss of physical autonomy and but also causes metabolic abnormalities that collectively lead to frailty and mortality in the aging. There is an urgent unmet medical need for appropriate therapeutic strategies of sarcopenia. Ginkgo biloba extract (GBE), a phytochemical product from ginkgo biloba leaf, exerted the therapeutic efficacy on many aging-related diseases, regarding Parkinson's disease, Alzheimer's disease and cerecardiovascular system diseases. However, whether GBE improves sarcopenia remains to be determined. Methods, Results & Conclusion GBE and Ginkgolide B, one unique component from GBE, were orally gavaged into ovariectomized (OVX) mice or 20 month-old female mice, and then the muscle mass and strength were measured after for treating two months. The role of Ginkgolide B was further tested in C2C12-derived myotubes. The effect of ginkgolide B on the expression of myogenic profile, differentiation index and fusion index of C2C12-derived myotubes were determined following by qPCR and myosin heavy chain staining. Our results showed that GBE was able to increase lean-to-body mass in OVX mice. We further demonstrated that ginkgolide B could improve sarcopenia in both OVX and aged mice, which is evidenced by the increased muscle mass and motor performances after treatment. Ginkgolide B promoted myogentic differentiation by improving bone-muscle crosstalk, and it also protected myotubes from glucocorticoid-induced muscle atrophy by regulating myostatin-to-ubiquitin proteasome system. In addition, ginkgolide B is able to enrich microvasculature in aged skeletal muscle that underpins appropriate muscular perfusion from local niche and circulation, hence restoring the loss of skeletal muscle mass and function during aging. Furthermore, we found that ginkgolide B reversed the expression of circulating pro-inflammatory cytokines in aged mice that may contribute to the improvement of sarcopenia. Collectively, our results demonstrated that ginkgolide B is sufficient to improve the debilitating sarcopenia and enlightens an affirmative prospect on treating sarcopenia.

Vasodilatory Actions of Glucagon-Like Peptide 1 Are Preserved in Skeletal and Cardiac Muscle Microvasculature but Not in Conduit Artery in Obese Humans With Vascular Insulin Resistance.

Obesity is associated with microvascular insulin resistance, which is characterized by impaired insulin-mediated microvascular recruitment. Glucagon-like peptide 1 (GLP-1) recruits skeletal and cardiac muscle microvasculature, and this action is preserved in insulin-resistant rodents. We aimed to examine whether GLP-1 recruits microvasculature and improves the action of insulin in obese humans. Fifteen obese adults received intravenous infusion of either saline or GLP-1 (1.2 pmol/kg/min) for 150 min with or without a euglycemic insulin clamp (1 mU/kg/min) superimposed over the last 120 min. Skeletal and cardiac muscle microvascular blood volume (MBV), flow velocity and blood flow, brachial artery diameter and blood flow, and pulse wave velocity (PWV) were determined. Insulin failed to change MBV or flow in either skeletal or cardiac muscle, confirming the presence of microvascular insulin resistance. GLP-1 infusion alone increased MBV by ∼30% and ∼40% in skeletal and cardiac muscle, respectively, with no change in flow velocity, leading to a significant increase in microvascular blood flow in both skeletal and cardiac muscle. Superimposition of insulin to GLP-1 infusion did not further increase MBV or flow in either skeletal or cardiac muscle but raised the steady-state glucose infusion rate by ∼20%. Insulin, GLP-1, and GLP-1 + insulin infusion did not alter brachial artery diameter and blood flow or PWV. The vasodilatory actions of GLP-1 are preserved in both skeletal and cardiac muscle microvasculature, which may contribute to improving metabolic insulin responses and cardiovascular outcomes. In obese humans with microvascular insulin resistance, GLP-1's vasodilatory actions are preserved in both skeletal and cardiac muscle microvasculature, which may contribute to improving metabolic insulin responses and cardiovascular outcomes.