Skeletal Muscle Arterioles Research Articles

Effect of myogenic tone on agonist-mediated vasoconstriction in isolated arteries: A computational study

Background and objectiveVasoconstriction of the resistance artery is mainly determined by an integrated action of multiple local stimuli acting on the vascular smooth muscle cells, which include neuronal delivery of α-adrenoceptor agonists and intraluminal pressure. The contractile activity of the arterial wall has been extensively studied ex vivo using isolated arterial preparations and myography techniques. However, agonist-mediated vasoconstriction response is often confounded by local effects of other stimuli (e.g., pressure) and, it remained unclear whether the pressure-induced myogenic response has any implication on the efficacy of agonist-mediated vasoconstriction during blood flow regulation in tissues. A quantitative understanding of the influence of each stimulus is necessary to understand the interaction between multiple regulatory mechanisms, which is required to ensure timely oxygen delivery to meet tissue needs. MethodsWe developed a simple empirical model of isolated vessel vasoreactivity that includes passive vessel wall mechanics and a lumped representation of active smooth muscle activation as a function of agonist concentration and pressure. Pressure myograph data in dog renal arterioles and rat femoral arterioles, isovolumic myograph data in rat femoral arteries, and vasoactive data in rat skeletal muscle arterioles were analyzed using the model. The effect of physiological pressure changes on the sensitivities of vascular segments to adrenergic agonists phenylephrine and norepinephrine was evaluated. ResultsModel-based analysis of isolated vasoreactivity data, obtained due to changes in pressure and vasoconstricting agonists revealed that the strength of myogenic response of a resistance vessel has a strong influence on the sensitivity and dynamics of agonist response. An increase in intraluminal pressure was found to reduce the magnitude of agonist-mediated tone by lowering the sensitivity of the vessel segment to agonist. The passive mechanical properties of arterial wall considearably influence the agonist-mediated contraction in isolated arteries. These results demonstrate how passive vessel wall mechanics may dominate the vasoactive responses of the common myogenic and adrenergic pathways of smooth muscle contraction in blood flow regulation, supporting a long standing notion that there exists segment-specific vasoregulation in microvascular networks of various tissues. ConclusionThe present model provides a simple and powerful tool for quantifying ex vivo vasoreactivity of asolated arteries to qualitatively study the interaction between myogenic and α-adrenergic control of vascular tone in isolated vessels. Analysis of pressure myography data and isovolumic myography data in different sizes of vessels and tissues, in response to norepinephrine and phenylephrine revealed the importance of passive vessel mechanics in arteriolar vasomotion and setting up of basal vasomotor tone at single vessel-level. The present study will be useful to quantify the extent to which myogenic tone may influence agonist-mediated vasoconstriction and agonist effect on pressure-mediated myogenic response in microvascular networks during blood flow regulation in tissues.

Abstract 58: The 5-HT7 receptor contributes to increased hindquarter blood flow caused by skeletal muscle contraction

Exogenous serotonin (5-hydroxytryptamine, 5-HT) causes a reproducible decrease in blood pressure in normotensive and hypertensive rats. This is associated with dilation of arterioles in skeletal muscle. The 5-HT7 receptor KO rat strain allowed us to test the role of this receptor in both responses as well as the effects of endogenous 5-HT -- acting at 5-HT7 receptors -- on normal circulatory regulation. First, we hypothesized that the 5-HT7 receptor KO rat would have a higher hindquarter (HQ) vascular resistance (HQVR) than wildtype (WT) because of loss of this receptor. In rats anesthetized with isoflurane, instrumented with arterial catheters and flow probes on the terminal aorta, male KO rats had significantly higher resting HQVR [mm Hg/ml/min; KO (16.0±2.0) vs WT (10.8±0.6.0), p = 0.04] and lower HQ blood flow than normal Sprague-Dawley (SD) rats. No differences in resting HQVR or HQ flow were observed in female rats. Intravenous infusion of 5-HT (50 ug/kg/min) reduced systemic blood pressure and HQVR; these parameters were significantly attenuated (~56%) in male KO rats versus WT and SD animals. In females, falls in systemic blood pressure and HQVR were absent in both KO and WT rats. Second, as 5-HT has been implicated in the pathophysiology of heart failure (HF), a left anterior descending artery ligation model of HF was used to investigate the potential role of 5-HT7 regulation of HQ flow in HF-associated exercise intolerance. The ability of hindlimb muscle stimulation to increase blood flow using transdermal neuromuscular electrical stimulation was tested (NMES). M and F KO rats showed a reduced ability to increase HQ flow during muscle contraction vs WT (% over basal: M WT = 118±18 vs KO =14.6±7.1; F WT= 101±12 vs KO = 7.6±6) rats. This was observed in both intact and HF rats (in which flow changes were already impaired). Acute administration of the 5-HT7 receptor antagonist SB269970 abolished the ability of NMES to increase HQ flow in normal SD rats. Importantly, the microcirculatory capacity, visualized by Lectin-594, was not significantly different in the gastrocnemius muscle of WT (arbitrary fluorescence units/area: 946±40) vs KO (914±69). These data support that the 5-HT7 receptor, likely activated by endogenous 5-HT, is an important regulator of skeletal muscle vascular resistance. Thus, 5-HT may have a larger role in normal and pathophysiological regulation of the circulation than has previously been appreciated.

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.

Regulation of Skeletal Muscle Resistance Arteriolar Tone: Integration of Multiple Mechanisms

Introduction: Physiological system complexity represents an imposing challenge to gaining insight into how arteriolar behavior emerges. Further, mechanistic complexity in arteriolar tone regulation requires that a systematic determination of how these processes interact to alter vascular diameter be undertaken. Methods: The present study evaluated the reactivity of ex vivo proximal and in situ distal resistance arterioles in skeletal muscle with challenges across the full range of multiple physiologically relevant stimuli and determined the stability of responses over progressive alterations to each other parameter. The five parameters chosen for examination were (1) metabolism (adenosine concentration), (2) adrenergic activation (norepinephrine concentration), (3) myogenic activation (intravascular pressure), (4) oxygen (superfusate PO₂), and (5) wall shear rate (altered intraluminal flow). Vasomotor tone of both arteriole groups following challenge with individual parameters was determined; subsequently, responses were determined following all two- and three-parameter combinations to gain deeper insight into how stimuli integrate to change arteriolar tone. A hierarchical ranking of stimulus significance for establishing arteriolar tone was performed using mathematical and statistical analyses in conjunction with machine learning methods. Results: Results were consistent across methods and indicated that metabolic and adrenergic influences were most robust and stable across all conditions. While the other parameters individually impact arteriolar tone, their impact can be readily overridden by the two dominant contributors. Conclusion: These data suggest that mechanisms regulating arteriolar tone are strongly affected by acute changes to the local environment and that ongoing investigation into how microvessels integrate stimuli regulating tone will provide a more thorough understanding of arteriolar behavior emergence across physiological and pathological states.

5-HT7 receptors mediate dilation of rat cremaster muscle arterioles in vivo.

Serotonin (5-HT) infusion in vivo causes hypotension and a fall in total peripheral resistance. However, the vascular segment and the receptors that mediate this response remain in question. We hypothesized that 5-HT7 receptors mediate arteriolar dilation to 5-HT in skeletal muscle microcirculation. Cremaster muscles of isoflurane-anesthetized male Sprague-Dawley rats were prepared for in vivo microscopy of third- and fourth-order arterioles and superfused with physiological salt solution at 34°C. Quantitative real-time PCR (RT-PCR) was applied to pooled samples of first- to third-order cremaster arterioles (2-4 rats/sample) to evaluate 5-HT7 receptor expression. Topical 5-HT (1-10 nmols) or the 5-HT1/7 receptor agonist, 5-carboxamidotryptamine (10-30 nM), dilated third- and fourth-order arterioles, responses that were abolished by 1 μM SB269970, a selective 5-HT7 receptor antagonist. In contrast, dilation induced by the muscarinic agonist, methacholine (100 nmols) was not inhibited by SB269970. Serotonin (10 nmols) failed to dilate cremaster arterioles in 5-HT7 receptor knockout rats whereas arterioles in wild-type litter mates dilated to 1 nmol 5-HT, a response blocked by 1 μM SB269970. Quantitative RT-PCR revealed that cremaster arterioles expressed mRNA for 5-HT7 receptors. 5-HT7 receptors mediate dilation of small arterioles in skeletal muscle and likely contribute to 5-HT-induced hypotension, in vivo.

Arteriolar dysgenesis in ischemic-regenerating skeletal muscle revealed by automated micromorphometry, computational modeling, and perfusion analysis.

Rebuilding the local vasculature is central to restoring the health of muscles subjected to ischemic injury. Arteriogenesis yields remodeled collateral arteries that circumvent the obstruction, and angiogenesis produces capillaries to perfuse the regenerating myofibers. However, the vital intervening network of arterioles that feed the regenerated capillaries is poorly understood and is an investigative challenge. We used machine learning and automated micromorphometry to quantify the arteriolar landscape in distal hindlimb muscles in mice that have regenerated after femoral artery excision. Assessment of 1,546 arteriolar sections revealed a striking (>2-fold) increase in arteriolar density in regenerated muscle 14 and 28 days after ischemic injury. Lumen caliber was initially similar to that of control arterioles but after 4 wk lumen area was reduced by 46%. In addition, the critical smooth muscle layer was attenuated throughout the arteriolar network, across a 150- to 5-µm diameter range. To understand the consequences of the reshaped distal hindlimb arterioles, we undertook computational flow modeling, which revealed blunted flow augmentation. Moreover, impaired flow reserve was confirmed in vivo by laser-Doppler analyses of flow in response to directly applied sodium nitroprusside. Thus, in hindlimb muscles regenerating after ischemic injury, the arteriolar network is amplified, inwardly remodels, and is diffusely undermuscularized. These defects and the associated flow restraints could contribute to the deleterious course of peripheral artery disease and merit attention when considering therapeutic innovations.NEW & NOTEWORTHY We report a digital pipeline for interrogating the landscape of arterioles in mouse skeletal muscle, using machine learning and automated micromorphometry. This revealed that in muscle regenerating after ischemic injury, the arteriolar density is increased but lumen caliber and smooth muscle content are reduced. Computational modeling and experimental validation reveal this arteriolar network to be functionally compromised, with diminished microvascular flow reserve.

5‐HT7 Receptors Mediate Dilation of Rat Cremaster Muscle Arterioles in vivo

Serotonin (5‐HT) infusion in vivo causes hypotension and a fall in total peripheral resistance (TPR) that is mediated by 5‐HT7 receptors. 5‐HT dilates small arterioles in skeletal muscle, a microcirculation that importantly contributes to TPR. However, the receptors that mediate this dilation have not been established. Therefore, we tested the hypothesis that 5‐HT7 receptors mediate the arteriolar dilation in the skeletal muscle microcirculation. Cremaster muscles of isoflurane‐anesthetized male Sprague‐Dawley rats were prepared for digital in vivo microscopy and superfused with physiological salt solution (PSS) at 34 °C. Bolus (1‐10 nmols) application of 5‐HT into the superfusate caused substantial dilation of 3rd to 5th order arterioles from 10 ± 1 to 27 ± 1 μm (n = 10 arterioles from 4 rats, p <0.0001) that recovered within 2‐3 min. Dilation to bolus application of 5‐HT was inhibited by superfusion with 1 μM SB269970, a selective 5‐HT7 receptor antagonist (diameter in SB = 9 ± 1 μm; diameter in SB + 5‐HT = 10 ± 1 μm; n = 10 arterioles from 4 rats; p = 0.8763). Similarly, steady‐state superfusion with 10‐30 nM of the 5‐HT1/7 receptor agonist 5‐carboxamidotryptamine (5‐CT) dilated 3rd‐5th‐order arterioles from 16 ± 3 to 37 ± 5 μm (n = 7 arterioles from 3 rats, p = 0.0004). This dilation was reversed (diameter in 1 μM SB269970 = 13 ± 2 μm, n = 7, p = 0.1479 vs. baseline) or blocked (diameter in 1 μM SB269970 + 10‐30 nM 5‐CT = 10 ± 2 μm, n = 7, p = 0.0005 vs. 5‐CT alone) by 1 μM SB269970. Dilation of arterioles to the muscarinic agonist, methacholine (MCH) was not inhibited by SB269970 verifying the specificity of the antagonist; arterioles dilated from 13 ± 2 μm to 25 ± 2 μm to 100 nmols MCH and dilated from 9 ± 2 μm to 32 ± 2 μm in response to MCH in the presence of 1 μM SB269970; n = 10 arterioles from 4 rats; p = 0.7232 vs. dilation in the absence of SB269970. Consistent with these data, pooled samples (4‐5 rats) of 1st and 2nd – order cremaster arterioles expressed message for 5‐HT7 receptors by quantitative real‐time PCR (CT = 36.4 ± 0.74, n = 3 pooled samples, p = 0.039 vs CT = 40). These data support our hypothesis that 5‐HT7 receptors mediate dilation of small arterioles in skeletal muscle and likely contribute to the observed hypotension when these receptors are engaged in vivo.

A theoretical model for flow regulation in the cerebral cortex: role of penetrating arterioles

Activation of a region of the brain results in a local increase in blood flow, a process known as neurovascular coupling (NVC). Here, a theoretical model of blood flow regulation in the cerebral cortex is utilized to investigate the potential role of penetrating arterioles as the primary mechanism for local control of blood flow. Penetrating arterioles connect pial arterioles with the capillary meshwork deeper in the cortex and have a typical length of approximately 600 μm. This is about one‐sixth of the length of corresponding small arterioles in skeletal muscle. The question arises whether these relatively short arterioles can achieve the range of flow modulation observed in the cerebral cortex. A previously developed compartmental model for flow regulation in skeletal muscle was modified to represent the structure of cerebral microvasculature. A compartment consisting of penetrating arterioles with a reference diameter of 14.8 μm and a length of 600 μm is assumed to provide active control of blood flow. Smooth muscle tone in these arterioles is assumed to depend on a combination of signals representing myogenic, shear‐dependent, and metabolic responses. The metabolic response is represented by a change in endothelial cell membrane potential, propagated upstream along arterioles by conducted responses. Sensitivity of diameter to membrane potential was determined based on experimental data. According to the model, a hyperpolarization of 40 mV can result in an increase in local blood flow by approximately 70%. Since increases in local blood flow of 50% with activation have been observed, these results imply that vasoconstriction and vasodilation by penetrating arterioles can provide typical levels of blood flow control seen in neurovascular coupling.

Role of Potassium Channel Function and Long‐term Exercise Training in Soleus Resistance Arterioles

IntroductionNuclear factor (erythroid‐derived 2)‐like 2 (Nrf2) expression has been linked to potassium (K+) channel function. We hypothesized that myogenic responses are enhanced by exercise training and also modulated by K+ channel function but would be impaired in arterioles from skeletal muscle of trained rats in which Nrf2 (Nrf2 KO) is deleted.MethodsExercise training was performed in male wildtype (WT) Sprague‐Dawley rats, and their littermates lacking the Nrf2 gene (Nrf2 KO). Rats were exercise trained using a mixed moderate and high‐intensity interval treadmill program for 10‐12 weeks. A WT cage‐confined control group was also studied to compare the impact of exercise training. After exercise training or sedentary cage‐confinement, soleus resistance arterioles were isolated and cannulated for the assessment of vascular reactivity. Vasodilatory responses to low concentrations of KCl [10 and 20mM} were assessed. Vasoconstrictor responses to higher concentrations of KCl [30–100mM] and myogenic responses to increasing intraluminal pressure (pressure 0cm H2O – 130cm H2O) were also assessed. The contribution of inward rectifying K+ channels (Kir) to vasodilation during low concentrations of KCl was assessed by treatment with barium chloride (BaCl2, 100mM).ResultsSoleus resistance arteriole myogenic responsiveness was reduced in trained Nrf2 KO as compared to WT (p<0.01). Trained WT rats produced a significantly higher myogenic responsiveness than sedentary WT controls (p<0.05). Vasodilation to low concentrations of KCl was reduced in soleus arterioles from trained Nrf2 KO rats as compared to arterioles from trained WT rats (p<0.01). Exercise training did not alter vasodilation to low KCl concentrations in WT rats (p>0.05). Constriction to higher concentrations of KCl was similar in arterioles from trained WT and Nrf2 KO rats. Inhibiting Kir channels with BaCl2 eliminated the genotype‐ and training‐related differences in myogenic responses and vasodilation to KCl.ConclusionOur data indicate that Nrf2 may be a critical regulator of vascular adaptation that occurs in skeletal muscle arterioles in response to long‐term exercise training. Elimination of differences in myogenic responsiveness by BaCl2 suggest that Nrf2‐mediated adaptation of the myogenic responsiveness may involve Kir channels.

Stimulation of the dorsolateral prefrontal cortex modulates muscle sympathetic nerve activity and blood pressure in humans.

IntroductionMuscle sympathetic nerve activity (MSNA) controls the diameter of arterioles in skeletalmuscle, contributing importantly to the beat-to-beat regulation of blood pressure (BP). Although brain imaging studies have shown that bursts of MSNA originate in the rostral ventrolateral medulla, other subcortical and cortical structures—including the dorsolateral prefrontal cortex (dlPFC)—contribute.HypothesisWe tested the hypothesis that MSNA and BP could be modulated by stimulating the dlPFC.MethoddlPFC. In 22 individuals MSNA was recorded via microelectrodes inserted into the common peroneal nerve, together with continuous BP, electrocardiographic, and respiration.Stimulation of the right (n=22) or left dlPFC (n=10) was achieved using transcranial alternating current (tcACS; +2 to −2mA, 0.08 Hz,100 cycles), applied between the nasion and electrodes over the F3 or F4 EEG sites on the scalp.ResultsSinusoidal stimulation of either dlPFC caused cyclicmodulation of MSNA, BP and heart rate, and a significant increase in BP.ConclusionWe have shown, for the first time, that tcACS of the dlPFC in awake humans causes partial entrainment of MSNA, heart rate and BP, arguing for an important role of this higher-level cortical area in the control of cardiovascular function.

Impaired microcirculatory function, mitochondrial respiration, and oxygen utilization in skeletal muscle of claudicating patients with peripheral artery disease.

Peripheral artery disease (PAD) is an atherosclerotic disease that impairs blood flow and muscle function in the lower limbs. A skeletal muscle myopathy characterized by mitochondrial dysfunction and oxidative damage is present in PAD; however, the underlying mechanisms are not well established. We investigated the impact of chronic ischemia on skeletal muscle microcirculatory function and its association with leg skeletal muscle mitochondrial function and oxygen delivery and utilization capacity in PAD. Gastrocnemius samples and arterioles were harvested from patients with PAD (n = 10) and age-matched controls (Con, n = 11). Endothelium-dependent and independent vasodilation was assessed in response to flow (30 μL·min-1), acetylcholine, and sodium nitroprusside (SNP). Skeletal muscle mitochondrial respiration was quantified by high-resolution respirometry, microvascular oxygen delivery, and utilization capacity (tissue oxygenation index, TOI) were assessed by near-infrared spectroscopy. Vasodilation was attenuated in PAD (P < 0.05) in response to acetylcholine (Con: 71.1 ± 11.1%, PAD: 45.7 ± 18.1%) and flow (Con: 46.6 ± 20.1%, PAD: 29.3 ± 10.5%) but not SNP (P = 0.30). Complex I + II state 3 respiration (P < 0.01) and TOI recovery rate were impaired in PAD (P < 0.05). Both flow and acetylcholine-mediated vasodilation were positively associated with complex I + II state 3 respiration (r = 0.5 and r = 0.5, respectively, P < 0.05). Flow-mediated vasodilation and complex I + II state 3 respiration were positively associated with TOI recovery rate (r = 0.8 and r = 0.7, respectively, P < 0.05). These findings suggest that chronic ischemia attenuates skeletal muscle arteriole endothelial function, which may be a key mediator for mitochondrial and microcirculatory dysfunction in the PAD leg skeletal muscle. Targeting microvascular dysfunction may be an effective strategy to prevent and/or reverse disease progression in PAD.NEW & NOTEWORTHY Ex vivo skeletal muscle arteriole endothelial function is impaired in claudicating patients with PAD, and this is associated with attenuated skeletal muscle mitochondrial respiration. In vivo skeletal muscle oxygen delivery and utilization capacity is compromised in PAD, and this may be due to microcirculatory and mitochondrial dysfunction. These results suggest that targeting skeletal muscle arteriole function may lead to improvements in skeletal muscle mitochondrial respiration and oxygen delivery and utilization capacity in claudicating patients with PAD.

TRPV1 in arteries enables a rapid myogenic tone.

Arterioles maintain blow flow by adjusting their diameter in response to changes in local blood pressure. In this process called the myogenic response, a vascular smooth muscle mechanosensor controls tone predominantly through altering the membrane potential. In general, myogenic responses occur slowly (minutes). In the heart and skeletal muscle, however, tone is activated rapidly (tens of seconds) and terminated by brief (100 ms) arterial constrictions. Previously, we identified extensive expression of TRPV1 in the smooth muscle of arterioles supplying skeletal muscle, heart and fat. Here we reveal a critical role for TRPV1 in the rapid myogenic tone of these tissues. TRPV1 antagonists dilated skeletal muscle arterioles in vitro and in vivo, increased coronary flow in isolated hearts, and transiently decreased blood pressure. All of these pharmacologic effects were abolished by genetic disruption of TRPV1. Stretch of isolated vascular smooth muscle cells or raised intravascular pressure in arteries triggered Ca2+ signalling and vasoconstriction. The majority of these stretch-responses were TRPV1-mediated, with the remaining tone being inhibited by the TRPM4 antagonist, 9-phenantrol. Notably, tone developed more quickly in arteries from wild-type compared with TRPV1-null mice. Furthermore, the immediate vasodilation following brief constriction of arterioles depended on TRPV1, consistent with a rapid deactivation of TRPV1. Pharmacologic experiments revealed that membrane stretch activates phospholipase C/protein kinase C signalling combined with heat to activate TRPV1, and in turn, L-type Ca2+ channels. These results suggest a critical role, for TRPV1 in the dynamic regulation of myogenic tone and blood flow in the heart and skeletal muscle. KEY POINTS: We explored the physiological role of TRPV1 in vascular smooth muscle. TRPV1 antagonists dilated skeletal muscle arterioles both ex vivo and in vivo, increased coronary perfusion and decreased systemic blood pressure. Stretch of arteriolar myocytes and increases in intraluminal pressure in arteries triggered rapid Ca2+ signalling and vasoconstriction respectively. Pharmacologic and/or genetic disruption of TRPV1 significantly inhibited the magnitude and rate of these responses. Furthermore, disrupting TRPV1 blunted the rapid vasodilation evoked by arterial constriction. Pharmacological experiments identified key roles for phospholipase C and protein kinase C, combined with temperature, in TRPV1-dependent arterial tone. These results show that TRPV1 in arteriolar myocytes dynamically regulates myogenic tone and blood flow in the heart and skeletal muscle.

Differential and Synergistic Effects of Low Birth Weight and Western Diet on Skeletal Muscle Vasculature, Mitochondrial Lipid Metabolism and Insulin Signaling in Male Guinea Pigs.

Low birth weight (LBW) offspring are at increased risk for developing insulin resistance, a key precursor in metabolic syndrome and type 2 diabetes mellitus. Altered skeletal muscle vasculature, extracellular matrix, amino acid and mitochondrial lipid metabolism, and insulin signaling are implicated in this pathogenesis. Using uteroplacental insufficiency (UPI) to induce intrauterine growth restriction (IUGR) and LBW in the guinea pig, we investigated the relationship between UPI-induced IUGR/LBW and later life skeletal muscle arteriole density, fibrosis, amino acid and mitochondrial lipid metabolism, markers of insulin signaling and glucose uptake, and how a postnatal high-fat, high-sugar “Western” diet (WD) modulates these changes. Muscle of 145-day-old male LBW glucose-tolerant offspring displayed diminished vessel density and altered acylcarnitine levels. Disrupted muscle insulin signaling despite maintained whole-body glucose homeostasis also occurred in both LBW and WD-fed male “lean” offspring. Additionally, postnatal WD unmasked LBW-induced impairment of mitochondrial lipid metabolism, as reflected by increased acylcarnitine accumulation. This study provides evidence that early markers of skeletal muscle metabolic dysfunction appear to be influenced by the in utero environment and interact with a high-fat/high-sugar postnatal environment to exacerbate altered mitochondrial lipid metabolism, promoting mitochondrial overload.

Survey of the extracellular matrix architecture across the rat arterial tree

ObjectiveTo understand arterial remodeling and the pathophysiology of arterial diseases, it is necessary to understand the baseline qualities and variations in arterial structure. Arteries could differ in wall thickness, laminar structure, and laminar fenestration depending on their position within the arterial tree. We endeavored to evaluate and compare the extracellular matrix structure of different arteries throughout the arterial tree, from the aorta to the adductor muscle arteriole, with a particular focus on the internal elastic lamina (IEL). MethodsArterial segments were harvested from male Sprague-Dawley rats and imaged using multiple modalities. En face scans by multiphoton microscopy were used to compare native-state adventitial collagen undulation and IEL fenestration. ResultsCollagen undulation was similar across most examined arteries but straighter in the skeletal muscle arterioles (P < .05). The elastic lamellae showed several differences. The IEL fenestrae were similar in average size among abdominal aorta and celiac, renal, common iliac, and common femoral arteries (range, 14-24 μm2), with wide within-vessel variance (square of the standard deviation, 462-1904 μm4). However, they tended to be smaller (9.08 μm2) and less variable (square of the standard deviation, 88.3 μm4) in the popliteal artery. Fenestrae were greater in number in the superior mesenteric artery (SMA; 6686/mm2; P < .05) and profunda femoris artery (PFA; 11,042/mm2; P < .05) compared with the other examined vessels, which ranged in surface density from 3143/mm2 to 4362/mm2. The SMA and PFA also showed greater total fenestration as a proportion of the IEL surface area (SMA, 15.04%; P < .05; PFA, 24.11%; P < .001) than the other examined arteries (range of means, 4.7%-9.4%). The arteriolar IEL was structurally distinct, comparable to a low-density wireframe. Other structural differences were also noted, including differences in the number of medial lamellae along the arterial tree. ConclusionsWe found that vessels at different locations along the arterial tree differ in structure. The SMA, PFA, and intramuscular arterioles have fundamental differences in the extracellular matrix structure compared with other arteries. Location-specific features such as the medial lamellae number and elastic laminar structure might have relevance to physiology and confer vulnerabilities to the development of pathology.

Microvascular Sex- and Age- Dependent Phosphodiesterase Expression

Objective: The cyclic nucleotide second messengers, cAMP and cGMP, are pivotal regulators of vascular functions; their cellular levels are tightly controlled by the cyclic nucleotide hydrolases, phosphodiesterases (PDE). Biologic sex and age are recognized as independent factors impacting the mechanisms mediating both vascular health and dysfunction. This study focused on microvessels isolated from male and female rats before (juvenile) and after (adult) sexual maturity under resting conditions. We tested the hypothesis that sexual dimorphism in microvascular PDE expression would be absent in juvenile rats, but would manifest in adult rats. Methods: Abdominal skeletal muscle arterioles and venules were isolated from age-matched juvenile and adult male and female rats under resting conditions. Transcripts of five PDE families (1–5) associated with coronary and vascular function with a total of ten genes were measured using TaqMan real-time RT-PCR and protein expression of microvessel PDE4 was assessed using immunoblotting and immunofluorescence. Results: Overall expression levels of PDE5A were highest while PDE3 levels were lowest among the five PDE families (p < 0.05) regardless of age or sex. Contrary to our hypothesis, in juveniles, sexual dimorphism in PDE expression was observed in three genes: arterioles (PDE1A, female > male) and venules (PDE1B and 3A, male > female). In adults, gene expression levels in males were higher than females for five genes in arterioles (PDE1C, 3A, 3B, 4B, 5A) and three genes (PDE3A, 3B, and 5A) in venules. Furthermore, age-related differences were observed in PDE1-5 (in males, adult > juvenile for most genes in arterioles; in females, adult > juvenile for arteriolar PDE3A; juvenile gene expression > adult for two genes in arterioles and three genes in venules). Immunoblotting and immunofluorescence analysis revealed protein expression of microvessel PDE4. Conclusion: This study revealed sexual dimorphism in both juvenile and adult rats, which is inconsistent with our hypothesis. The sex- and age-dependent differences in PDE expression implicate different modulations of cAMP and cGMP pathways for microvessels in health. The implication of these sex- and age-dependent differences, as well as the duration and microdomain of PDE1-5 activities in skeletal muscle microvessels, in both health and disease, require further investigation.

TRPV1 expressed throughout the arterial circulation regulates vasoconstriction and blood pressure.

The functional roles of the capsaicin receptor, TRPV1, outside of sensory nerves are unclear. We mapped TRPV1 in the mouse circulation, revealing extensive expression in the smooth muscle of resistance arterioles supplying skeletal muscle, heart and adipose tissue. Activation of TRPV1 in vascular myocytes constricted arteries, reduced coronary flow in isolated hearts and increased systemic blood pressure. These functional effects were retained after sensory nerve ablation, indicating specific signalling by arterial TRPV1. TRPV1 mediated the vasoconstrictive and blood pressure responses to the endogenous inflammatory lipid lysophosphatidic acid. These results show that TRPV1 in arteriolar myocytes modulates regional blood flow and systemic blood pressure, and suggest that TRPV1 may be a target of vasoactive inflammatory mediators. The capsaicin receptor, TRPV1, is a key ion channel involved in inflammatory pain signalling. Although mainly studied in sensory nerves, there are reports of TRPV1 expression in isolated segments of the vasculature, but whether the channel localizes to vascular endothelium or smooth muscle is controversial and the distribution and functional roles of TRPV1 in arteries remain unknown. We mapped functional TRPV1 expression throughout the mouse arterial circulation. Analysis of reporter mouse lines TRPV1PLAP-nlacZ and TRPV1-Cre:tdTomato combined with Ca2+ imaging revealed specific localization of TRPV1 to smooth muscle of terminal arterioles in the heart, adipose tissue and skeletal muscle. Capsaicin evoked inward currents (current density ∼10% of sensory neurons) and raised intracellular Ca2+ levels in arterial smooth muscle cells, constricted arterioles ex vivo and in vivo and increased systemic blood pressure in mice and rats. Further, capsaicin markedly and dose-dependently reduced coronary flow. Pharmacological and/or genetic disruption of TRPV1 abolished all these effects of capsaicin as well as vasoconstriction triggered by lysophosphatidic acid, a bioactive lipid generated by platelets and atherogenic plaques. Notably, ablation of sensory nerves did not affect the responses to capsaicin revealing a vascular smooth muscle-restricted signalling mechanism. Moreover, unlike in sensory nerves, TRPV1 function in arteries was resistant to activity-induced desensitization. Thus, TRPV1 activation in vascular myocytes enables a persistent depolarizing current, leading to constriction of coronary, skeletal muscle and adipose arterioles and a sustained increase in systemic blood pressure.

The Effects of Prolonged Mechanical Ventilation on Structural and Material Properties of Diaphragm Arterioles

IntroductionIn skeletal muscle arterioles, cellular and vasoactive responses are influenced by the physiochemical milieu of the surrounding tissue. Understanding the mechanical properties of the blood vessel wall yields insight into vascular function in health and disease. In blood vessels, the endothelium mediates flow‐dependent vasodilation and contributes to the regulation of arterial mechanics. Prolonged mechanical ventilation (MV)(&gt;6 h) results in large, time‐dependent reductions in diaphragmatic blood flow and impaired endothelium‐dependent (and ‐independent) vasodilation of first‐order (1A) medial costal diaphragm arterioles. The purpose of this investigation was to determine if the severe reductions in diaphragmatic blood flow (and shear‐stress) with MV impair the structural and material properties (i.e. increased stress/stretch relation and/or circumferential stretch) of 1A arterioles from the medial costal diaphragm.MethodsFemale Sprague‐Dawley rats (~5 mo) were randomly divided into two groups, spontaneous breathing (SB, n = 6) and prolonged MV (6 h MV, n =5). Following SB and 6 h MV, 1A medial costal diaphragm arterioles (~200 μm diameter, ~2 mm in length) were isolated and cannulated. After cannulation, vessels were adjusted to a fixed length (L, 1.5 mm) and pressurized. Vessels that exhibited no leaks were subjected to a step‐wise passive‐pressure diameter test (0–140 cmH2O) in calcium‐free Ringer’s solution, at a single fixed axial length (L). Inner diameter (μm) and wall thickness (μm) were measured at each pressure step and used to calculate wall: lumen ratio, incremental distensibility (%/mmHg), Cauchy‐stress (μ), and circumferential stretch (λθ).ResultsFollowing 6 h MV, in the undeformed condition (0 mmHg) the wall: lumen ratio was significantly smaller compared to SB (1.04 ±0.21vs. 2.60 ±0.40; p&lt;0.05). There were no differences in incremental distensibility (%/mmHg) and the stress/stretch (μvs λθ) relation between groups (p&gt;0.05). At every pressure step, circumferential stretch (λθ; undeformed radius (r)/current deformed radius (R)) was significantly increased with 6 h MV versus SB (p&lt;0.05).ConclusionDespite no change in the distensibility and structural behavior (stress/stretch) of resistance vessels from the medial costal diaphragm, prolonged MV resulted in altered material properties (i.e. elevated circumferential stretch (λθ)) of medial costal diaphragm arterioles. Increased circumferential stretch can enhance superoxide generation in the endothelium and vascular smooth muscle cells. Therefore, elevated circumferential stretch of 1A diaphragm arterioles in combination with the severe reductions in blood flow may, in part, explain the previously demonstrated vascular dysfunction in the diaphragm following MV.Support or Funding InformationSupported by: NIH HL137156‐01A1

Clinically‐Tested SOD Mimic, MnTnBuOE‐2‐PyP5+, Acutely Decreases Blood Pressure via Sympathoinhibition and Vasodilation

It is well‐established that reactive oxygen species (ROS), particularly superoxide (O2•−), produced in the vasculature and brain contribute to the pathogenesis of hypertension. We, and others, have previously reported that in vivo scavenging of O2·− via overexpression of superoxide dismutase (SOD) protein or SOD mimics decreases blood pressure in hypertensive animals. While few SOD mimics have transitioned to clinical trials, MnTnBuOE‐2‐PyP5+ (BuOE), a manganese porphyrin SOD mimic, is currently in clinical trials for normal tissue protection in cancer patients exposed to radiation therapy, and thus, provides hope for the use of SOD mimics in the clinical setting. Considering the extensive evidence indicating that scavenging of O2•− decreases hypertensive blood pressures, we tested the hypothesis that the SOD mimic, BuOE, decreases blood pressure in normotensive and angiotensin II (AngII)‐induced hypertensive mice. First, using electron paramagnetic resonance (EPR) spectroscopy and hypoxanthine (HX) + xanthine oxidase (XO) to generate O2•− in a cell‐free system, we confirmed the ability of BuOE to scavenge O2•−. Superoxide produced by HX + XO yielded an EPR spectrum amplitude of 5.6×106 ± 1.3×105 EPR arbitrary units (au), which was significantly (p &lt; 0.05) attenuated by 50 nM BuOE (3.3×106 ± 4.2×104 au). In normotensive and AngII‐induced hypertensive mice, BuOE (1 mg/kg, IP), significantly (p&lt;0.05) decreased mean arterial pressure (MAP), as measured by radiotelemetry, within 25 minutes post‐injection (normotensive ⊗ MAP: vehicle −3.3 ± 2.8; BuOE −27 ± 5.4 mmHg; hypertensive ⊗ MAP: vehicle 3.1 ± 7.2; BuOE −60 ± 14 mmHg). To explore the mechanism(s) by which BuOE decreases MAP, renal sympathetic nerve activity (RSNA) was recorded in normotensive rats injected with BuOE (0.1 mg/kg, IV). Immediately following BuOE injection, RSNA significantly (p &lt; 0.01) decreased by 35 ± 7.0% of baseline, followed by a significant (p &lt; 0.01) decrease in MAP (46 ± 13 mmHg). Additionally, to explore the impact of BuOE on vascular reactivity, video myography was performed on isolated skeletal muscle arterioles from normotensive rats. BuOE (2 μM) induced significant (p &lt; 0.05) vasodilation (31 ± 10%) that was attenuated by pretreating arterioles with the endothelial nitric oxide synthase (eNOS) inhibitor, L‐NAME (1 mM); thus, suggesting that enhanced nitric oxide bioavailability mediates BuOE‐induced vasodilation. Collectively, these data indicate that BuOE, a SOD mimic currently in clinical trials in cancer patients, acutely decreases blood pressure in both normotensive and hypertensive conditions by decreasing sympathetic nerve activity and inducing vasodilation.Support or Funding InformationUniversity of Nebraska Medical Center Graduate Student Assistantship/Fellowship

Type II Diabetes Mellitus in the Goto‐Kakizaki Rat Impairs Microvascular Function and Contributes to Premature Skeletal Muscle Fatigue

Despite extensive investigation into the impact of metabolic disease on vascular function and, by extension, tissue perfusion and organ function, interpreting results for specific risk factors can be complicated by the additional risks present in most models. To specifically determine the impact of type II diabetes without obesity on skeletal muscle microvascular structure/function, and on active hyperemia with elevated metabolic demand, we used 17‐week old Goto‐Kakizaki rats (GK) to study microvascular function at multiple levels of resolution. Gracilis muscle arterioles demonstrated blunted dilation to acetylcholine (both ex vivo proximal and in situ distal arterioles) and elevated shear (distal arterioles only). All other alterations to reactivity appeared to reflect compromised endothelial function associated with increased TxA2 production and oxidant stress/inflammation rather than alterations to vascular smooth muscle function. Structural changes to the microcirculation of GK were confined to reduced microvessel density of ~12%, with no evidence for altered vascular wall mechanics. Active hyperemia with either field stimulation of in situ cremaster muscle or electrical stimulation via the sciatic nerve for in situ gastrocnemius muscle was blunted in GK; primarily due to blunted functional dilation of skeletal muscle arterioles. The blunted active hyperemia was associated with impaired oxygen uptake (VO2) across the muscle and an accelerated muscle fatigue. Acute interventions to reduce oxidant stress (TEMPOL) and TxA2 action (SQ‐29548) or production (dazmegrel) improved muscle perfusion, VO2, and muscle performance. These results suggest that T2DM in GK impairs skeletal muscle arteriolar function apparently early in the progression of the disease and potentially via an increased ROS/inflammation‐induced TxA2 production/action on network function as a major contributing mechanism.Support or Funding InformationAmerican Heart Association and National Institutes of Health (USA); Canadian Institutes for Health Research and Natural Sciences and Engineering Research Council (Canada)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Type 2 diabetes mellitus in the Goto-Kakizaki rat impairs microvascular function and contributes to premature skeletal muscle fatigue.

Despite extensive investigation into the impact of metabolic disease on vascular function and, by extension, tissue perfusion and organ function, interpreting results for specific risk factors can be complicated by the additional risks present in most models. To specifically determine the impact of type 2 diabetes without obesity on skeletal muscle microvascular structure/function and on active hyperemia with elevated metabolic demand, we used 17-wk-old Goto-Kakizaki (GK) rats to study microvascular function at multiple levels of resolution. Gracilis muscle arterioles demonstrated blunted dilation to acetylcholine (both ex vivo proximal and in situ distal arterioles) and elevated shear (distal arterioles only). All other alterations to reactivity appeared to reflect compromised endothelial function associated with increased thromboxane (Tx)A2 production and oxidant stress/inflammation rather than alterations to vascular smooth muscle function. Structural changes to the microcirculation of GK rats were confined to reduced microvessel density of ~12%, with no evidence for altered vascular wall mechanics. Active hyperemia with either field stimulation of in situ cremaster muscle or electrical stimulation via the sciatic nerve for in situ gastrocnemius muscle was blunted in GK rats, primarily because of blunted functional dilation of skeletal muscle arterioles. The blunted active hyperemia was associated with impaired oxygen uptake (V̇o2) across the muscle and accelerated muscle fatigue. Acute interventions to reduce oxidant stress (TEMPOL) and TxA2 action (SQ-29548) or production (dazmegrel) improved muscle perfusion, V̇o2, and muscle performance. These results suggest that type 2 diabetes mellitus in GK rats impairs skeletal muscle arteriolar function apparently early in the progression of the disease and potentially via an increased reactive oxygen species/inflammation-induced TxA2 production/action on network function as a major contributing mechanism. NEW & NOTEWORTHY The impact of type 2 diabetes mellitus on vascular structure/function remains an area lacking clarity. Using diabetic Goto-Kakizaki rats before the development of other risk factors, we determined alterations to vascular structure/function and skeletal muscle active hyperemia. Type 2 diabetes mellitus reduced arteriolar endothelium-dependent dilation associated with increased thromboxane A2 generation. Although modest microvascular rarefaction was evident, there were no other alterations to vascular structure/function. Skeletal muscle active hyperemia was blunted, although it improved after antioxidant or anti-thromboxane A2 treatment.