The role of pannexin/purinergic signaling in intervascular communication from capillaries during skeletal muscle contraction in male Golden hamsters.

We sought to determine the physiological relevance of pannexin/purinergic-dependent signaling in mediating conducted vasodilation elicited by capillary stimulation through skeletal muscle contraction. Using hamster cremaster muscle and intravital microscopy we stimulated capillaries through local muscle contraction while observing the associated upstream arteriole. Capillaries were stimulated with muscle contraction at low and high contraction (6 and 60CPM) and stimulus frequencies (4 and 40 Hz) in the absence and presence of pannexin blocker mefloquine (MEF; 10-5 M), purinergic receptor antagonist suramin (SUR 10-5 M) and gap-junction uncoupler halothane (HALO, 0.07%) applied between the capillary stimulation site and the upstream arteriolar observation site. Conducted vasodilations elicited at 6CPM were inhibited by HALO while vasodilations at 60CPM were inhibited by MEF and SUR. The conducted response elicited at 4 Hz was inhibited by MEF while the vasodilation at 40 Hz was unaffected by any blocker. Therefore, upstream vasodilations resulting from capillary stimulation via muscle contraction are dependent upon a pannexin/purinergic-dependent pathway that appears to be stimulation parameter-dependent. Our data highlight a physiological importance of the pannexin/purinergic pathway in facilitating communication between capillaries and upstream arteriolar microvasculature and, consequently, indicating that this pathway may play a crucial role in regulating blood flow in response to skeletal muscle contraction.

Ergogenic effect of ischemic preconditioning is not directly conferred to isolated skeletal muscle via blood.

PURPOSE: Ischemic preconditioning (IPC) in humans has been demonstrated to confer ergogenic benefit to aerobic exercise performance, with an improvement in the response rate when the IPC stimulus is combined with concurrent exercise. Despite potential performance improvements, the nature of the neuronal and humoral mechanisms of conferral and their respective contributions to ergogenic benefit remain unclear. We sought to examine the effects of the humoral component of ischemic preconditioning on skeletal muscle tissue using preconditioned human serum and isolated mouse soleus. METHODS: Isolated mouse soleus was electrically stimulated to contract while in human serum preconditioned with either traditional (IPC) or augmented (AUG) ischemic preconditioning compared to control (CON) and exercise (ERG) preconditioning. Force frequency (FF) curves, twitch responses, and a fatigue-recovery protocol were performed on muscles before and after the addition of serum. After preconditioning, human participants performed a 4 km cycling time trial in order to identify responders and non-responders to IPC. RESULTS: No differences in indices of contractile function, fatiguability, nor recovery were observed between conditions in mouse soleus muscles. Further, no human participants improved performance in a 4-km cycling time trial in response to traditional nor augmented ischemic preconditioning compared to control or exercise conditions (CON 407.7 ± 41.1 s, IPC 411.6 ± 41.9 s, ERG 408.8 ± 41.4 s, AUG 414.1 ± 41.9 s). CONCLUSIONS: Our findings do not support the conferral of ergogenic benefit via a humoral component of IPC at the intracellular level. Ischemic preconditioning may not manifest prominently at submaximal exercise intensities, and augmented ischemic preconditioning may have a hormetic relationship with performance improvements.

Capillary communication: the role of capillaries in sensing the tissue environment, coordinating the microvascular, and controlling blood flow.

Historically, capillaries have been viewed as the microvascular site for flux of nutrients to cells and removal of waste products. Capillaries are the most numerous blood vessel segment within the tissue, whose vascular wall consists of only a single layer of endothelial cells and are situated within microns of each cell of the tissue, all of which optimizes capillaries for the exchange of nutrients between the blood compartment and the interstitial space of tissues. There is, however, a growing body of evidence to support that capillaries play an important role in sensing the tissue environment, coordinating microvascular network responses, and controlling blood flow. Much of our growing understanding of capillaries stems from work in skeletal muscle and more recent work in the brain, where capillaries can be stimulated by products released from cells of the tissue during increased activity and are able to communicate with upstream and downstream vascular segments, enabling capillaries to sense the activity levels of the tissue and send signals to the microvascular network to coordinate the blood flow response. This review will focus on the emerging role that capillaries play in communication between cells of the tissue and the vascular network required to direct blood flow to active cells in skeletal muscle and the brain. We will also highlight the emerging central role that disruptions in capillary communication may play in blood flow dysregulation, pathophysiology, and disease.

Dietary nitrate increases submaximal SERCA activity and ADP transfer to mitochondria in slow-twitch muscle of female mice.

Rapid oscillations in cytosolic calcium (Ca2+) coordinate muscle contraction, relaxation, and physical movement. Intriguingly, dietary nitrate decreases ATP cost of contraction, increases force production, and increases cytosolic Ca2+, which would seemingly necessitate a greater demand for sarcoplasmic reticulum Ca2+ ATPase (SERCA) to sequester Ca2+ within the sarcoplasmic reticulum (SR) during relaxation. As SERCA is highly regulated, we aimed to determine the effect of 7-day nitrate supplementation (1 mM via drinking water) on SERCA enzymatic properties and the functional interaction between SERCA and mitochondrial oxidative phosphorylation. In soleus, we report that dietary nitrate increased force production across all stimulation frequencies tested, and throughout a 25 min fatigue protocol. Mice supplemented with nitrate also displayed an ∼25% increase in submaximal SERCA activity and SERCA efficiency (P = 0.053) in the soleus. To examine a possible link between ATP consumption and production, we established a methodology coupling SERCA and mitochondria in permeabilized muscle fibers. The premise of this experiment is that the addition of Ca2+ in the presence of ATP generates ADP from SERCA to support mitochondrial respiration. Similar to submaximal SERCA activity, mitochondrial respiration supported by SERCA-derived ADP was increased by ∼20% following nitrate in red gastrocnemius. This effect was fully attenuated by the SERCA inhibitor cyclopiazonic acid and was not attributed to differences in mitochondrial oxidative capacity, ADP sensitivity, protein content, or reactive oxygen species emission. Overall, these findings suggest that improvements in submaximal SERCA kinetics may contribute to the effects of nitrate on force production during fatigue.NEW & NOTEWORTHY We show that nitrate supplementation increased force production during fatigue and increased submaximal SERCA activity. This was also evident regarding the high-energy phosphate transfer from SERCA to mitochondria, as nitrate increased mitochondrial respiration supported by SERCA-derived ADP. Surprisingly, these observations were only apparent in muscle primarily expressing type I (soleus) but not type II fibers (EDL). These findings suggest that alterations in SERCA properties are a possible mechanism in which nitrate increases force during fatiguing contractions.

Capillaries communicate with the arteriolar microvascular network by a pannexin/purinergic-dependent pathway in hamster skeletal muscle.

We sought to determine if a pannexin/purinergic-dependent intravascular communication pathway exists in skeletal muscle microvasculature that facilitates capillary communication with upstream arterioles that control their perfusion. Using the hamster cremaster muscle and intravital microscopy, we locally stimulated capillaries and observed the vasodilatory response in the associated upstream 4A arteriole. We stimulated capillaries with vasodilators relevant to muscle contraction: 10-6 M S-nitroso-N-acetyl-dl-penicillamine (SNAP; nitric oxide donor), 10-6 M adenosine, 10 mM potassium chloride, 10-5 M pinacidil, as well as a known initiator of gap-junction-dependent intravascular communication, acetylcholine (10-5 M), in the absence and the presence of the purinergic membrane receptor blocker suramin (10-5 M), pannexin blocker mefloquine (2 × 10-5 M), or probenecid (5 × 10-6 M) and gap-junction inhibitor halothane (0.07%) applied in the transmission pathway, between the capillary stimulation site and the upstream 4A observation site. Potassium chloride, SNAP, and adenosine-induced upstream vasodilations were significantly inhibited by suramin, mefloquine, and probenecid but not halothane, indicating the involvement of a pannexin/purinergic-dependent signaling pathway. Conversely, SNAP-induced upstream vasodilation was only inhibited by halothane indicating that communication was facilitated by gap junctions. Both pinacidil and acetylcholine were inhibited by suramin but only acetylcholine was inhibited by halothane. These data demonstrate the presence of a pannexin/purinergic-dependent communication pathway between capillaries and upstream arterioles controlling their perfusion. This pathway adds to the gap-junction-dependent pathway that exists at this vascular level as well. Given that vasodilators relevant to muscle contraction can use both of these pathways, our data implicate the involvement of both pathways in the coordination of skeletal muscle blood flow.NEW & NOTEWORTHY Blood flow control during increased metabolic demand in skeletal muscle is not fully understood. Capillaries have been implicated in controlling blood flow to active skeletal muscle, but how capillaries communicate to the arteriolar vascular network is not clear. Our study uncovers a novel pathway through which capillaries can communicate to upstream arterioles to cause vasodilation and therefore control perfusion. This work implicates a new vascular communication pathway in blood flow control in skeletal muscle.

Do skeletal muscle motor units and microvascular units align to help match blood flow to metabolic demand?

PURPOSE: We explore the motor unit recruitment and control of perfusion of microvascular units in skeletal muscle to determine whether they coordinate to match blood flow to metabolic demand. METHODS: The PubMed database was searched for historical, current and relevant literature. RESULTS: A microvascular, or capillary unit consists of 2-20 individual capillaries. Individual capillaries within a capillary unit cannot increase perfusion independently of other capillaries within the unit. Capillary units perfuse a short segment of approx. 12 muscle fibres located beside each other. Motor units consist of muscle fibres that can be dispersed widely within the muscle volume. During a contraction, where not all motor units are recruited, muscle fibre contraction will result in increased perfusion of associated capillaries as well as all capillaries within that capillary unit. Perfusion of the entire capillary unit will result in an increased blood flow delivery to muscle fibres associated with active motor unit plus approximately 11 other inactive muscle fibres within the same region. This will result in an overperfusion of the muscle resulting in blood flow in excess of the muscle fibre needs. CONCLUSIONS: Given the architecture of the capillary units and the dispersed nature of muscle fibres within a motor unit, during submaximal contractions, where not all motor units are recruited, there will be a greater perfusion to the muscle than that predicted by the number of active muscle fibres. Such overperfusion brings into question if blood flow and metabolic demand are as tightly matched as previously assumed.

Incorporating higher order thinking and deep learning in a large, lecture-based human physiology course: can we do it?

Large classes taught with didactic lectures and assessed with multiple-choice tests are commonly reported to promote lower order (LO) thinking and a surface approach (SA) to learning. Using a case study design, we hypothesized that incorporating instructional scaffolding of core physiology principles and assessing students exclusively with long-answer written tests would encourage higher order (HO) thinking and promote a deep approach (DA) to learning in a two-course physiology sequence (Phys I and II), despite their large size. Test questions were categorized as LO or HO according to the Blooming Biology Tool, and students' LO and HO performance was determined for each of six tests across the two courses. The validated Revised Two-Factor Study Process Questionnaire survey tool was administered at the beginning and end of each course to measure student approach to learning. HO performance was maintained across Phys I (72.9 ± 19.4 vs. 74.8 ± 20.7%, P = 0.37) and significantly improved across Phys II (69.9 ± 18.4 vs. 79.4 ± 14.8%, P < 0.001). Unexpectedly, students' LO performance declined from the beginning to end of Phys I (78.5 ± 20.6 vs. 69.4 ± 17.9%, P < 0.001) and Phys II (80.5 ± 19.6 vs. 72.2 ± 24.3%, P < 0.001). Students' approach to learning did not change throughout Phys I or II, but at each time point students preferred a DA over a SA. Taken together, these results indicate that an intentionally designed large lecture class can support a DA to learning and suggests that this teaching and assessment structure may be particularly well suited to promote HO thinking, albeit possibly at the expense of LO thinking.

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.

Splenic blood-flow response following myocardial infarction in rat.

During physiological stress (e.g., exercise, hypoxia), blood flow is shunted to specific anatomical regions to protect critical organs; yet, splenic blood flow in these circumstances remains to be investigated. Despite being classically viewed as a non-critical organ, recent experimental and epidemiological evidence suggests the spleen plays a significant role in cardiovascular pathophysiology. We hypothesized that splenic blood flow is prioritized in the development of heart failure (i.e., chronic state of reduced cardiac output). Five-week-old male Wistar rats were randomized for either myocardial infarction (MI; n = 58) or sham (n = 56) surgery. At 2, 5, and 9 weeks post-surgery, Doppler ultrasound measurements of the splenic, left renal, left common carotid, and left femoral arteries were performed. Cardiac function was assessed at all time points using echocardiography and at 9 weeks post-surgery using invasive hemodynamic analysis. Splenic and cerebral blood flow was preferentially maintained at 9 weeks post-MI, whereas blood flow to the lower limb and kidney were reduced. Spleen size increased by 5 weeks post-MI and remained elevated. Splenic blood flow was maintained in conditions of decreased cardiac output, when other tissues showed decreased blood flow. The maintenance of blood flow in the face of decreased cardiac output indicates that splenic function is being prioritized during heart failure.

Arteriolar and capillary responses to CO2 and H+ in hamster skeletal muscle microvasculature: Implications for active hyperemia.

OBJECTIVE: We hypothesized that CO2 and H+ stimulate capillaries and arterioles to produce local and conducted vasodilations required to coordinate the distribution of blood flow to contracting skeletal muscle fibers. METHODS: CO2 and H+ independently and in combination were applied to 2A arterioles (first branch order from the 1A feed arteriole) and capillaries of the in situ, blood-perfused hamster cremaster muscle. The resulting local and conducted vasodilations were measured. RESULTS: H+ (pH: 7.2-6.6) and CO2 (5% and 10%) applied to the vascular network induced 2A arteriolar vasodilations, while 15% CO2 produced vasoconstriction. Localized application of H+ produced 2A arteriolar vasodilation, while 15% CO2 resulted in a variable response. Simultaneous application of CO2 and H+ did not result in the predicted additive effects. Application of CO2 and H+ alone or combined on arterioles or capillaries did not induce a conducted response. CONCLUSIONS: CO2 and H+ produce arteriolar vasodilation but, critically, cannot stimulate the spread of vasodilation throughout the network, thus limiting their ability to coordinating blood flow to contracting skeletal muscle fibers. Given their potential for interaction, the importance of CO2 and H+ may lie in their ability to modify the effects of other vasodilators.

Capillary response to skeletal muscle contraction: evidence that redundancy between vasodilators is physiologically relevant during active hyperaemia.

KEY POINTS: The current theory behind matching blood flow to metabolic demand of skeletal muscle suggests redundant interactions between metabolic vasodilators. Capillaries play an important role in blood flow control given their ability to respond to muscle contraction by causing conducted vasodilatation in upstream arterioles that control their perfusion. We sought to determine whether redundancies occur between vasodilators at the level of the capillary by stimulating the capillaries with muscle contraction and vasodilators relevant to muscle contraction. We identified redundancies between potassium and both adenosine and nitric oxide, between nitric oxide and potassium, and between adenosine and both potassium and nitric oxide. During muscle contraction, we demonstrate redundancies between potassium and nitric oxide as well as between potassium and adenosine. Our data show that redundancy is physiologically relevant and involved in the coordination of the vasodilator response during muscle contraction at the level of the capillaries. ABSTRACT: We sought to determine if redundancy between vasodilators is physiologically relevant during active hyperaemia. As inhibitory interactions between vasodilators are indicative of redundancy, we tested whether vasodilators implicated in mediating active hyperaemia (potassium (K+ ), adenosine (ADO) and nitric oxide (NO)) inhibit one another's vasodilatory effects through direct application of pharmacological agents and during muscle contraction. Using the hamster cremaster muscle and intravital microscopy, we locally stimulated capillaries with one vasodilator in the absence and the presence of a second vasodilator (10-7 m S-nitroso-N-acetylpenicillamine (SNAP), 10-7 m ADO, 10 mm KCl) applied sequentially and simultaneously, and observed the response in the associated upstream 4A arteriole controlling the perfusion of the stimulated capillary. We found that KCl significantly attenuated SNAP- and ADO-induced vasodilatations by ∼49.7% and ∼128.0% respectively and ADO significantly attenuated KCl- and SNAP-induced vasodilatations by ∼94.7% and ∼59.6%, respectively. NO significantly attenuated KCl vasodilatation by 93.8%. Further, during muscle contraction we found that inhibition of NO production using l-NG -nitroarginine methyl ester and inhibition of ADO receptors using xanthine amine congener was effective at inhibiting contraction-induced vasodilatation but only in the presence of K+ release channel inhibition. Thus, only when the inhibiting vasodilator K+ was blocked was the second vasodilator, NO or ADO, able to produce effective vasodilatation. Therefore, we show that there are inhibitory interactions between specific vasodilators at the level of the capillary. Further, these inhibitions can be observed during muscle contraction indicating that redundancies between vasodilators are physiologically relevant and influence vasodilatation during active hyperaemia.

Central-acting therapeutics alleviate respiratory weakness caused by heart failure-induced ventilatory overdrive.

Diaphragmatic weakness is a feature of heart failure (HF) associated with dyspnea and exertional fatigue. Most studies have focused on advanced stages of HF, leaving the cause unresolved. The long-standing theory is that pulmonary edema imposes a mechanical stress, resulting in diaphragmatic remodeling, but stable HF patients rarely exhibit pulmonary edema. We investigated how diaphragmatic weakness develops in two mouse models of pressure overload-induced HF. As in HF patients, both models had increased eupneic respiratory pressures and ventilatory drive. Despite the absence of pulmonary edema, diaphragmatic strength progressively declined during pressure overload; this decline correlated with a reduction in diaphragm cross-sectional area and preceded evidence of muscle weakness. We uncovered a functional codependence between angiotensin II and ß-adrenergic (ß-ADR) signaling, which increased ventilatory drive. Chronic overdrive was associated with increased PERK (double-stranded RNA-activated protein kinase R-like ER kinase) expression and phosphorylation of EIF2α (eukaryotic translation initiation factor 2α), which inhibits protein synthesis. Inhibition of ß-ADR signaling after application of pressure overload normalized diaphragm strength, Perk expression, EIF2α phosphorylation, and diaphragmatic cross-sectional area. Only drugs that were able to penetrate the blood-brain barrier were effective in treating ventilatory overdrive and preventing diaphragmatic atrophy. These data provide insight into why similar drugs have different benefits on mortality and symptomatology, despite comparable cardiovascular effects.

Dose-dependent inhibition of uterine contractility by nitric oxide: A potential mechanism underlying persistent breeding-induced endometritis in the mare.

Nitric oxide (NO) may have a role in persistent breeding-induced endometritis in mares through an inhibitory effect on uterine contractility. The objectives of this study were to test the effect of NO on spontaneous uterine contractility in-vitro and to evaluate whether this effect varied between the longitudinal and circular muscle layers of the uterus. Reproductive tracts were collected from eight euthanized non-pregnant mares (age 4-19 years; body weight 405-530 kg). Transrectal examination of the reproductive tract was performed before euthanasia to evaluate stage of the estrous cycle and presence of any apparent abnormality. After euthanasia, one uterine tissue sample was collected for histological evaluation and four full-thickness uterine tissue strips (10-12 mm × 2 mm), two parallel to each muscle layer, were excised for in-vitro contractility evaluation. Strips were suspended in tissue chambers containing Krebs-Henseleit solution, with continuous aeration (95% O2-5% CO2; pH 7.4) at 37 °C. After equilibration, spontaneous contractility was recorded (pre-treatment) and strips excised in each direction were randomly allocated to each of two groups: 1) SNAP (S-nitroso-N-acetylpenicillamine, an NO donor); or 2) NAP (N-acetyl-d-penicillamine, vehicle and time-matched control). These were treated at 15 min intervals with increasing concentrations (10-7 M to 10-3 M) of SNAP and NAP, respectively. Contractility data was recorded throughout the experiment. An interaction effect of group-by-concentration was observed (P < 0.0001). The mean contractility after treatment with 10-4 M and 10-3 M SNAP were significantly lower than the pre-treatment contractility and the mean contractility after treatment with lower SNAP concentrations. In contrast, contractility did not change significantly in the NAP treated controls. The effect of treatment on uterine contractility was not influenced by age or weight of the mare, stage of estrous cycle, uterine histology grade, or muscle layer. Secondary findings included significant main effects of stage of estrous cycle (increased contractility in estrus compared to diestrus), uterine histology grade (decreased contractility in grade IIB compared to grade I) and age (decreased contractility in mares aged > 8 years compared to mares aged ≤ 8 years). In conclusion, results of this study indicate that NO has a dose-dependent inhibitory effect on spontaneous uterine contractility irrespective of the muscle layer in the mare.

Capillary endothelial cells as coordinators of skeletal muscle blood flow during active hyperemia.

In this invited review, we explore the burgeoning possibility of capillary endothelial cells as coordinators of skeletal muscle blood flow in response to muscle contraction. The idea that the capillary is an active vascular unit in skeletal muscle microcirculation starkly diverges from the traditional dogma that seats arterioles as the central controllers of blood flow during exercise. This review aims to incite discussion as we revisit and rethink the role of capillary endothelial cells in skeletal muscle. We discuss the potential for a mismatch in the architectural relationships between the arteriolar microvasculature and contracting motor units that would negate consistent communication between them. We review the data from the past two decades demonstrating that capillaries are ideally located architecturally to communicate with skeletal muscle fibers and are mechanistically capable of signaling upstream arterioles that control their own perfusion. We show that the orchestration of a coordinated vascular response necessary to support active skeletal muscle fibers cannot be achieved by the arterioles, but rather it is the capillaries that drive the blood flow response to muscle contraction. Thus, capillaries need to be seriously considered as critical in the coordination of skeletal muscle blood flow during active hyperemia.

Potassium inhibits nitric oxide and adenosine arteriolar vasodilatation via K(IR) and Na(+)/K(+) ATPase: implications for redundancy in active hyperaemia.

Redundancy, in active hyperaemia, where one vasodilator can compensate for another if the first is missing, would require that one vasodilator inhibits the effects of another; therefore, if the first vasodilator is inhibited, its inhibitory influence on the second vasodilator is removed and the second vasodilator exerts a greater vasodilatory effect. We aimed to determine whether vasodilators relevant to skeletal muscle contraction [potassium chloride (KCl), adenosine (ADO) and nitric oxide] inhibit one another and, in addition, to investigate the mechanisms for this interaction. We used the hamster cremaster muscle and intravital microscopy to directly visualize 2A arterioles when exposed to a range of concentrations of one vasodilator [10(-8) to 10(-5) M S-nitroso-N-acetyl penicillamine (SNAP), 10(-8) to 10(-5) M ADO, 10 and 20 mM KCl] in the absence and then in the presence of a second vasodilator (10(-7) M ADO, 10(-7) M SNAP, 10 mM KCl). We found that KCl significantly attenuated SNAP-induced vasodilatations by ∼65.8% and vasodilatations induced by 10(-8) to 10(-6) M ADO by ∼72.8%. Furthermore, we observed that inhibition of KCl vasodilatation, by antagonizing either Na(+)/K(+) ATPase using ouabain or inward rectifying potassium channels using barium chloride, could restore the SNAP-induced vasodilatation by up to ∼53.9% and 30.6%, respectively, and also restore the ADO-induced vasodilatations by up to ∼107% and 76.7%, respectively. Our data show that vasodilators relevant to muscle contraction can interact in a way that alters the effectiveness of other vasodilators. These data suggest that active hyperaemia may be the result of complex interactions between multiple vasodilators via a redundant control paradigm.

Respiratory muscle weakness in the Zucker diabetic fatty rat.

The obesity epidemic is considered one of the most serious public health problems of the modern world. Physical therapy is the most accessible form of treatment; however, compliance is a major obstacle due to exercise intolerance and dyspnea. Respiratory muscle atrophy is a cause of dyspnea, yet little is known of obesity-induced respiratory muscle dysfunction. Our objective was to investigate whether obesity-induced skeletal muscle wasting occurs in the diaphragm, the main skeletal muscle involved in inspiration, using the Zucker diabetic fatty (ZDF) rat. After 14 wk, ZDF rats developed obesity, hyperglycemia, and insulin resistance, compared with lean controls. Hemodynamic analysis revealed ZDF rats have impaired cardiac relaxation (P = 0.001) with elevated end-diastolic pressure (P = 0.006), indicative of diastolic dysfunction. Assessment of diaphragm function revealed weakness (P = 0.0296) in the absence of intrinsic muscle impairment in ZDF rats. Diaphragm morphology revealed increased fibrosis (P < 0.0001), atrophy (P < 0.0001), and reduced myosin heavy-chain content (P < 0.001), compared with lean controls. These changes are accompanied by activation of the myostatin signaling pathway with increased serum myostatin (P = 0.017), increased gene expression (P = 0.030) in the diaphragm and retroperitoneal adipose (P = 0.033), and increased SMAD2 phosphorylation in the diaphragm (P = 0.048). Here, we have confirmed the presence of respiratory muscle atrophy and weakness in an obese, diabetic model. We have also identified a pathological role for myostatin signaling in obesity, with systemic contributions from the adipose tissue, a nonskeletal muscle source. These findings have significant implications for future treatment strategies of exercise intolerance in an obese, diabetic population.

Local control of blood flow during active hyperaemia: what kinds of integration are important?

The focus of this review is on local mechanisms modifying arteriolar resistance to match blood flow to metabolism. In skeletal muscle many local mediators are known, including K(+) , nitric oxide (NO), purines and prostaglandins. Each accounts for about 30% of the response; it is widely held that these act redundantly: this concept awaits systematic testing. Understanding signal integration also requires consideration of microvascular network morphology in relation to local communication pathways between endothelial and smooth muscle cells (which are critical for many local responses, including dilatation to skeletal muscle contraction) and in relation to the spread of vasodilator signals up- and downstream throughout the network. Mechanisms mediating the spread of dilatation from local to remote sites have been well studied using acetylcholine (ACh), but remote dilatations to contraction of skeletal muscle fibres also occur. Importantly, these mechanisms clearly differ from those initiated by ACh, but much remains undefined. Furthermore, capillaries contribute to metabolic dilatation as they dilate arterioles directly upstream in response to vasoactive agents or contraction of adjacent muscle fibres. Given the dispersed arrangement of motor units, precise matching of flow to metabolism is not attainable unless signals are initiated only by 'active' capillaries. As motor units are recruited, signals that direct blood flow towards these active fibres will eventually be supported by local and spreading responses in the arterioles associated with those fibres. Thus, mechanisms of integration of vasodilator signalling across elements of the microvasculature remain an important area of focus for new studies.

Prostaglandins induce vasodilatation of the microvasculature during muscle contraction and induce vasodilatation independent of adenosine.

Blood flow data from contracting muscle in humans indicates that adenosine (ADO) stimulates the production of nitric oxide (NO) and vasodilating prostaglandins (PG) to produce arteriolar vasodilatation in a redundant fashion such that when one is inhibited the other can compensate. We sought to determine whether these redundant mechanisms are employed at the microvascular level. First, we determined whether PGs were involved in active hyperaemia at the microvascular level. We stimulated four to five skeletal muscle fibres in the anaesthetized hamster cremaster preparation in situ and measured the change in diameter of 2A arterioles (maximum diameter 40 µm, third arteriolar level up from the capillaries) at a site of overlap with the stimulated muscle fibres before and after 2 min of contraction [stimulus frequencies: 4, 20 and 60 Hz at 15 contractions per minute (CPM) or contraction frequencies of 6, 15 or 60 CPM at 20 Hz; 250 ms train duration]. Muscle fibres were stimulated in the absence and presence of the phospholipase A2 inhibitor quinacrine. Further, we applied a range of concentrations of ADO (10(-7)-10(-5) M) extraluminally, (to mimic muscle contraction) in the absence and presence of L-NAME (NO synthase inhibitor), indomethacin (INDO, cyclooxygenase inhibitor) and L-NAME + INDO and observed the response of 2A arterioles. We repeated the latter experiment on a different level of the cremaster microvasculature (1A arterioles) and on the microvasculature of a different skeletal muscle (gluteus maximus, 2A arterioles). We observed that quinacrine inhibited vasodilatation during muscle contraction at intermediate and high contraction frequencies (15 and 60 CPM). L-NAME, INDO and L-NAME + INDO were not effective at inhibiting vasodilatation induced by any concentration of ADO tested in 2A and 1A arterioles in the cremaster muscle or 2A arterioles in the gluteus maximus muscle. Our data show that PGs are involved in the vasodilatation of the microvasculature in response to muscle contraction but did not obtain evidence that extraluminal ADO causes vasodilatation through NO or PG or both. Thus, we propose that PG-induced microvascular vasodilation during exercise is independent of ADO.

Gestational modification of murine spiral arteries does not reduce their drug-induced vasoconstrictive responses in vivo.

Dynamic control of maternal blood flow to the placenta is critical for healthy pregnancy. In many tissues, microvasculature arteries control the flow. The uterine/endometrial vascular bed changes during pregnancy include physiological remodeling of spiral arteries from constricted artery-like structures to dilated vein-like structures between Gestation Day 8 (gd8) and gd12 in mice and wk 12-16 in humans. These changes occur, in part, due to local environmental changes such as decidualization, recruitment of maternal uterine natural killer cells, and invasion of conceptus-derived trophoblasts. No current preparations permit in vivo testing of decidual microvascular reactivity. We report an in vivo intravital fluorescence microscopy model that permits functional study of the entire uterine microvascular bed (uterine, arcuate, radial, basal, and spiral arteries) in gravid C57BL/6 mice. Vascular reactivities were measured at gd8 prespiral arterial remodeling and gd12 (postremodeling) to a range of concentrations of adenosine (10(-8)-10(-6) M), acetylcholine (10(-7)-10(-5) M), phenylephrine (10(-7)-10(-5) M), and angiotensin II (10(-8)-10(-6) M). At baseline, each arterial branch order was significantly more dilated on gd12 than gd8. Each microvascular level responded to each agonist on gd8 and gd12. At gd12, vasodilation to adenosine was attenuated in uterine, arcuate, and basal arteries, while constrictor activity to angiotensin II was enhanced in uterine and arcuate arteries. The tendency for increasing vasoconstriction between gd8 to gd12 and the constrictor responses of modified spiral arteries were unexpected findings that may reflect influences of the intact in vivo environment rather than inherent properties of the vessels and may be relevant to ongoing human pregnancies.