Exaggerated cardiovascular responses to treadmill running in rats with peripheral arterial insufficiency.

Patients with atherosclerotic peripheral artery disease have an augmented pressor response to treadmill walking, but the underlying mechanisms remain poorly understood and difficult to isolate because of the confounding presence of numerous cardiovascular risk factors. In the present study, we tested the hypothesis that a chronic deficit in muscle blood flow capacity would be sufficient to trigger an exaggerated pressor response to dynamic exercise. Sprague-Dawley rats (5 male and 5 female) were instrumented with radiotelemetry devices to measure the cardiovascular responses to treadmill running before and after bilateral femoral artery ligation, which has been previously shown to reduce the blood flow capacity of distal hindlimb muscles by >60%. Treadmill running evoked reproducible increases in mean arterial pressure (MAP) and heart rate (HR), which were significantly augmented 3 days after femoral artery ligation in both male rats [ΔMAP: +10 ± 1 (SE) vs. +18 ± 3 mmHg and ΔHR: +94 ± 12 vs. +148 ± 15 beats/min, P < 0.05] and female rats (ΔMAP: +16 ± 3 vs. +30 ± 5 mmHg and ΔHR: +128 ± 20 vs. +178 ± 19 beats/min, P < 0.05). Similar exaggerated MAP and HR responses were observed at repeated intervals between 3 and 65 days postligation. These findings indicate that a chronic deficit in muscle blood flow capacity is an important, persistent cause of the abnormal pressor and cardioaccelerator responses to dynamic exercise in both male and female rats with peripheral arterial insufficiency. NEW & NOTEWORTHY Using radiotelemetry to assess cardiovascular effects of exercise, we showed that femoral artery obstruction in male and female rats is an important, persistent cause of exaggerated pressor and cardioaccelerator responses to treadmill running. This translational model reproduces the abnormal cardiovascular response to exercise seen in patients with peripheral artery disease. Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/treadmill-bp-in-simulated-peripheral-artery-disease/ .

The role played by oxidative stress in evoking the exercise pressor reflex in health and simulated peripheral artery disease.

KEY POINTS: Ligating the femoral artery of a rat for 72 h, a model for peripheral artery disease, causes an exaggerated exercise pressor reflex in response to muscle contraction. Likewise, the hindlimb muscles of rats with ligated femoral arteries show increased levels of reactive oxygen species. Infusion of tiron, a superoxide scavenger, attenuated the exaggerated pressor reflex and reduced reactive oxygen species production in rats with ligated femoral arteries. Conversely, we found no effect of tiron infusion on the pressor reflex in rats with patent femoral arteries. These results suggest a role of reactive oxygen species with respect to causing the exaggerated pressor response to contraction seen in rats with ligated arteries and peripheral artery disease. ABSTRACT: Contraction of muscle evokes the exercise pressor reflex (EPR), which is expressed partly by increases in heart rate and arterial pressure. Patients with peripheral artery disease (PAD) show an exaggerated EPR, sometimes report pain when walking and are at risk for cardiac arrthymias. Previous research suggested that reactive oxygen species (ROS) mediate the exaggerated EPR associated with PAD. To examine the effects of ROS on the EPR, we infused a superoxide scavenger, tiron, into the superficial epigastric artery of decerebrated rats. In some, we simulated PAD by ligating a femoral artery for 72 h before the experiment. The peak EPR in 'ligated' rats during saline infusion averaged 31 ± 4 mmHg, whereas the peak EPR in these rats during tiron infusion averaged 13 ± 2 mmHg (n = 12; P < 0.001); the attenuating effect of tiron on the EPR was partly reversed when saline was reinfused into the superficial epigastric artery (21 ± 2 mmHg; P < 0.01 vs. tiron). The peak EPR in 'ligated' rats was also attenuated (n = 7; P < 0.01) by infusion of gp91ds-tat, a peptide that blocks the activity of NAD(P)H oxidase. Tiron infusion had no effect on the EPR in rats with patent femoral arteries (n = 9). Western blots showed that the triceps surae muscles of 'ligated' rats expressed more Nox2 and p67phox, which are components of NADPH oxidase, compared to triceps surae muscles of 'freely perfused' rats. Tiron added to muscle homogenates reduced ROS production in vitro. The results of the present study provide further evidence indicating that ROS mediates the exaggeration of EPR in rats with simulated PAD.

Dual AAV therapy ameliorates exercise-induced muscle injury and functional ischemia in murine models of Duchenne muscular dystrophy.

Neuronal nitric oxide synthase (nNOS) membrane delocalization contributes to the pathogenesis of Duchenne muscular dystrophy (DMD) by promoting functional muscle ischemia and exacerbating muscle injury during exercise. We have previously shown that supra-physiological expression of nNOS-binding mini-dystrophin restores normal blood flow regulation and prevents functional ischemia in transgenic mdx mice, a DMD model. A critical next issue is whether systemic dual adeno-associated virus (AAV) gene therapy can restore nNOS-binding mini-dystrophin expression and mitigate muscle activity-related functional ischemia and injury. Here, we performed systemic gene transfer in mdx and mdx4cv mice using a pair of dual AAV vectors that expressed a 6 kb nNOS-binding mini-dystrophin gene. Vectors were packaged in tyrosine mutant AAV-9 and co-injected (5 × 10(12) viral genome particles/vector/mouse) via the tail vein to 1-month-old dystrophin-null mice. Four months later, we observed 30-50% mini-dystrophin positive myofibers in limb muscles. Treatment ameliorated histopathology, increased muscle force and protected against eccentric contraction-induced injury. Importantly, dual AAV therapy successfully prevented chronic exercise-induced muscle force drop. Doppler hemodynamic assay further showed that therapy attenuated adrenergic vasoconstriction in contracting muscle. Our results suggest that partial transduction can still ameliorate nNOS delocalization-associated functional deficiency. Further evaluation of nNOS binding mini-dystrophin dual AAV vectors is warranted in dystrophic dogs and eventually in human patients.

Oxidative stress and enhanced sympathetic vasoconstriction in contracting muscles of nitrate-tolerant rats and humans.

Sympathetic vasoconstriction is normally attenuated in exercising muscle, but this functional sympatholysis is impaired in rats with hypertension or heart failure due to elevated levels of reactive oxygen species (ROS) in muscle. Whether ROS have a similar effect in the absence of cardiovascular disease or whether these findings extend to humans is not known. We therefore tested the hypothesis that chronic treatment with nitroglycerin (NTG) to induce nitrate tolerance, which is associated with excessive ROS production, impairs functional sympatholysis in healthy rats and humans. NTG treatment increased ethidium fluorescence in rat muscles and urinary F(2)-isoprostanes in humans, demonstrating oxidative stress. In vehicle-treated rats, sympathetic nerve stimulation (1 to 5 Hz) evoked decreases in femoral vascular conductance at rest (range, -30 to -63%) that were attenuated during hindlimb contraction (range, -2 to -31%; P < 0.05). In NTG-treated rats, vasoconstrictor responses were similar at rest, but were enhanced during contraction (range, -17 to -50%; P < 0.05 vs. vehicle). Infusion of the ROS scavenger tempol restored sympatholysis in these rats. In humans, reflex sympathetic activation during lower body negative pressure (LBNP) evoked decreases in muscle oxygenation in resting forearm (-12 ± 1%) that were attenuated during handgrip exercise (-3 ± 1%; P < 0.05). When these subjects became nitrate tolerant, LBNP-induced decreases in muscle oxygenation were unaffected at rest, but were enhanced during exercise (-9 ± 1%; P < 0.05 vs. before NTG). Collectively, these data indicate that functional sympatholysis is impaired in otherwise healthy nitrate-tolerant rats and humans by a mechanism probably involving muscle oxidative stress.

Functional sympatholysis is impaired in hypertensive humans.

In healthy individuals, sympathetic vasoconstriction is markedly blunted in exercising muscles to optimize blood flow to the metabolically active muscle fibres. This protective mechanism, termed functional sympatholysis, is impaired in rat models of angiotensin-dependent hypertension. However, the relevance of these findings to human hypertension is unknown. Therefore, in 13 hypertensive and 17 normotensive subjects we measured muscle oxygenation and forearm blood flow (FBF) responses to reflex increases in sympathetic nerve activity (SNA) evoked by lower body negative pressure (LBNP) at rest and during moderate-intensity rhythmic handgrip exercise. In the normotensives, LBNP caused decreases in oxygenation and FBF (−16 ± 2% and −23 ± 4%, respectively) in resting forearm but not in exercising forearm (−1 ± 2% and −1 ± 3%, respectively; P < 0.05 vs. rest). In the hypertensives, LBNP evoked decreases in oxygenation and FBF that were similar in the resting and exercising forearm (−14 ± 2% vs. −12 ± 2% and −20 ± 3% vs. −13 ± 2%, respectively; P > 0.05), indicating impaired functional sympatholysis. In the hypertensives, SNA was unexpectedly increased by 54 ± 11% during handgrip alone. However, when SNA was experimentally increased during exercise in the normotensives, sympatholysis was unaffected. Treatment for 4 weeks with the angiotensin receptor blocker irbesartan, but not with the thiazide-type diuretic chlorthalidone, restored sympatholysis in the hypertensives. These data provide the first evidence that functional sympatholysis is impaired in hypertensive humans by a mechanism that appears to involve an angiotensin-dependent increase in sympathetic vasoconstriction in the exercising muscles.

Neural control of the circulation.

The purpose of this brief review is to highlight key concepts about the neural control of the circulation that graduate and medical students should be expected to incorporate into their general knowledge of human physiology. The focus is largely on the sympathetic nerves, which have a dominant role in cardiovascular control due to their effects to increase cardiac rate and contractility, cause constriction of arteries and veins, cause release of adrenal catecholamines, and activate the renin-angiotensin-aldosterone system. These effects, as well as the control of sympathetic outflow by the vasomotor center in the medulla and the importance of sensory feedback in the form of peripheral reflexes, especially the baroreflexes, are discussed in the context of cardiovascular regulation.

Inhibition of nitric oxide synthase evokes central sympatho-excitation in healthy humans.

Animal studies have indicated that nitric oxide is a key signalling molecule involved in the tonic restraint of central sympathetic outflow from the brainstem. Extension of these findings to humans has been difficult because systemic infusion of nitric oxide synthase (NOS) inhibitors increases blood pressure due to inhibition of endothelial NOS, resulting in activation of the arterial baroreflex and subsequent inhibition of central sympathetic outflow. To overcome this confounding inhibitory influence of the baroreflex, in the current study we directly measured skin sympathetic nerve activity (SNA), which is not under baroreceptor control. Healthy, normotensive humans were studied before, during a 60 min intravenous infusion of the NOS inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME; 4 mg kg(1)), and for 120 min following the infusion (i.e. 180 min total). Skin SNA and arterial blood pressure (BP) were continuously measured. BP was increased from baseline at the end of the l-NAME infusion (14 +/- 2 mmHg; P < 0.05) and remained significantly elevated for the remainder of the experiment (18 +/- 3 mmHg; P < 0.05). Similarly, systemic NOS inhibition produced time-dependent increases in skin SNA, such that skin SNA was elevated at the end of the l-NAME infusion (total activity, 200 +/- 22% baseline; P = 0.08) and was further increased at the end of the study protocol (total activity, 350 +/- 41% baseline; P < 0.05). Importantly, skin SNA remained unchanged during time and hypertensive (phenylephrine) control experiments. These findings indicate that pharmacological inhibition of NOS causes sympathetic activation and support a role of nitric oxide in central sympathetic control in humans.

C-reactive protein causes downregulation of vascular angiotensin subtype 2 receptors and systolic hypertension in mice.

BACKGROUND: Chronic elevations in circulating C-reactive protein (CRP) are associated with a greater risk of hypertension. Whether elevations in CRP cause hypertension is unknown. METHODS AND RESULTS: Chronic, conscious blood pressure (BP) measurements were performed by radiotelemetry in wild-type CF1 control and CF1 transgenic mice expressing rabbit CRP (CF1-CRP) under the regulation of the phosphoenolpyruvate carboxykinase promoter. Compared with controls, CF1-CRP mice had hypertension that was predominantly systolic, and the severity of hypertension varied in parallel with changes in CRP levels modulated by dietary manipulation. Mice that were hemizygous for the transgene with CRP levels of 9 microg/mL were also hypertensive, indicating that modest elevations in CRP are sufficient to alter BP. CRP transgenic mice had exaggerated BP elevation in response to angiotensin II and a reduction in vascular angiotensin receptor subtype 2 (AT2) expression. In contrast, the decline in BP with angiotensin receptor subtype 1 (AT1) antagonism and vascular AT1 abundance were unaltered, which indicates a selective effect of CRP on AT2. Ex vivo experiments further showed that the CRP-induced decrease in AT2 is a direct effect on the vascular wall, not requiring systemic responses, and that it is reversed by an NO donor, which indicates a role for NO deficiency in the process. In parallel, the chronic inhibition of NO synthase in wild-type mice attenuated vascular AT2 expression without affecting AT1. CONCLUSIONS: These findings provide direct evidence for CRP-induced hypertension, and they further identify a novel underlying mechanism involving downregulation of AT2 related to NO deficiency.

FcgammaRIIB mediates C-reactive protein inhibition of endothelial NO synthase.

C-reactive protein (CRP) is an acute-phase reactant that is positively correlated with cardiovascular disease risk and endothelial dysfunction. Whether CRP has direct actions on endothelium and the mechanisms underlying such actions are unknown. Here we show in cultured endothelium that CRP prevents endothelial NO synthase (eNOS) activation by diverse agonists, resulting in the promotion of monocyte adhesion. CRP antagonism of eNOS occurs nongenomically and is attributable to blunted eNOS phosphorylation at Ser1179. Okadaic acid or knockdown of PP2A by short-interference RNA reverses CRP antagonism of eNOS, indicating a key role for the phosphatase. Aggregated IgG, the known ligand for Fcgamma receptors, causes parallel okadaic acid-sensitive loss of eNOS function, FcgammaRIIB expression is demonstrable in endothelium, and heterologous expression studies reveal that CRP antagonism of eNOS requires FcgammaRIIB. In FcgammaRIIB(+/+) mice, CRP blunts acetylcholine-induced increases in carotid artery vascular conductance; in contrast, CRP enhances acetylcholine responses in FcgammaRIIB(-/-) mice. Thus FcgammaRIIB mediates CRP inhibition of eNOS via PP2A, providing a mechanistic link between CRP and endothelial dysfunction.

Skin blood flow influences near-infrared spectroscopy-derived measurements of tissue oxygenation during heat stress.

Near-infrared (NIR) spectroscopy is a noninvasive optical technique that is increasingly used to assess muscle oxygenation during exercise with the assumption that the contribution of skin blood flow to the NIR signal is minor or nonexistent. We tested this assumption in humans by monitoring forearm tissue oxygenation during selective cutaneous vasodilation induced by locally applied heat (n = 6) or indirect whole body heating (i.e., heating subject but not area surrounding NIR probes; n = 8). Neither perturbation has been shown to cause a measurable change in muscle blood flow or metabolism. Local heating (approximately 41 degrees C) caused large increases in the NIR-derived tissue oxygenation signal [before heating = 0.82 +/- 0.89 optical density (OD), after heating = 18.21 +/- 2.44 OD; P < 0.001]. Similarly, whole body heating (increase internal temperature 0.9 degrees C) also caused large increases in the tissue oxygenation signal (before heating = -0.31 +/- 1.47 OD, after heating = 12.48 +/- 1.82 OD; P < 0.001). These increases in the tissue oxygenation signal were closely correlated with increases in skin blood flow during both local heating (mean r = 0.95 +/- 0.02) and whole body heating (mean r = 0.89 +/- 0.04). These data suggest that the contribution of skin blood flow to NIR measurements of tissue oxygenation can be significant, potentially confounding interpretation of the NIR-derived signal during conditions where both skin and muscle blood flows are elevated concomitantly (e.g., high-intensity and/or prolonged exercise).

Vasomodulation by skeletal muscle-derived nitric oxide requires alpha-syntrophin-mediated sarcolemmal localization of neuronal Nitric oxide synthase.

Neuronal nitric oxide synthase (nNOS) is abundantly expressed in skeletal muscle where it associates with the dystrophin complex at the sarcolemma by binding to the PDZ domain of alpha-syntrophin. Nitric oxide (NO) produced by skeletal muscle nNOS is proposed to regulate blood flow in exercising muscle by diffusing from the skeletal muscle fibers to the nearby microvessels where it attenuates alpha-adrenergic vasoconstriction. In the present study, we hypothesized that sarcolemmal localization of nNOS is a critical determinant of the vasoregulatory effect of skeletal muscle-derived NO. To test this hypothesis, we performed experiments in alpha-syntrophin null mice and in transgenic mice expressing a mutated alpha-syntrophin lacking the PDZ domain (DeltaPDZ), both of which are characterized by reduced sarcolemmal nNOS. We found that modulation of alpha-adrenergic vasoconstriction was greatly impaired in the contracting muscles of the alpha-syntrophin null mice and transgenic DeltaPDZ mice compared with wild-type mice and transgenic mice expressing full-length alpha-syntrophin. These in vivo mouse studies highlight the functional importance of appropriate membrane targeting of nNOS by the dystrophin-associated protein alpha-syntrophin and may have implications for the development of potential gene therapy strategies to treat muscular dystrophy or other muscle-related diseases.

Nitric oxide pathway as new drug targets for refractory hypertension.

Nitric oxide (NO) is thought to reduce blood pressure by evoking vasodilation either directly by causing relaxation of vascular smooth muscle or indirectly by acting in the rostral brainstem to reduce central sympathetic outflow, which decreases the release of norepinephrine from sympathetic nerve terminals. An increasingly large body of literature suggests that alterations in the NO system may play an important role in the development or maintenance of clinical hypertension. As proof of concept, pharmacological inhibition of nitric oxide synthase (NOS) in humans and animals causes moderate to severe hypertension. Certain forms of secondary hypertension are accompanied by the accumulation of endogenous NOS inhibitors, which may contribute to the development of hypertension. Furthermore, targeted disruption of the endothelial isoform of NOS in mice causes moderate hypertension, implying that hypertension may also develop from reductions in NOS expression. These gene knockout studies in animals have initiated the search for single nucleotide polymorphisms in human NOS genes, which could potentially lead to decreases in NOS protein expression. Conversely, increases in NOS expression or NO production have been linked with several commonly used cardiovascular therapies, including exercise training and the use of both statins and angiotensin-converting enzyme inhibitors. Finally, increases in the production of oxidants such as superoxide anion can lead to the inactivation of NO, thereby reducing NO bioavailability. Thus, alterations in the expression or activity of NOS or in the availability of NO have the potential to play a causal role in clinical hypertension. The purpose of this article is to show how emerging basic research on the NO pathway is elucidating novel antihypertensive drug targets that are on the cusp of clinical application.

Functional sympatholysis in hypertension.

Sympathetic vasoconstriction is normally attenuated in exercising muscle by local changes in muscle metabolites and other substances that reduce vascular responsiveness to α-adrenergic receptor activation. Termed functional sympatholysis, this protective mechanism is thought to optimize muscle blood flow distribution to match perfusion with metabolic demand. Emerging evidence from both animal and human studies indicate that functional sympatholysis is impaired in hypertension and may constitute an important underlying cause of skeletal muscle malperfusion during exercise in this common cardiovascular condition. Findings from studies of animal models of hypertension and patients with essential hypertension will be integrated in this review to provide insight into the underlying mechanisms responsible for inappropriate sympathetic vasoconstriction in exercising muscle and the treatment options that may restore functional sympatholysis and improve muscle perfusion during exercise.

Neural control of muscle blood flow during exercise.

Activation of skeletal muscle fibers by somatic nerves results in vasodilation and functional hyperemia. Sympathetic nerve activity is integral to vasoconstriction and the maintenance of arterial blood pressure. Thus the interaction between somatic and sympathetic neuroeffector pathways underlies blood flow control to skeletal muscle during exercise. Muscle blood flow increases in proportion to the intensity of activity despite concomitant increases in sympathetic neural discharge to the active muscles, indicating a reduced responsiveness to sympathetic activation. However, increased sympathetic nerve activity can restrict blood flow to active muscles to maintain arterial blood pressure. In this brief review, we highlight recent advances in our understanding of the neural control of the circulation in exercising muscle by focusing on two main topics: 1) the role of motor unit recruitment and muscle fiber activation in generating vasodilator signals and 2) the nature of interaction between sympathetic vasoconstriction and functional vasodilation that occurs throughout the resistance network. Understanding how these control systems interact to govern muscle blood flow during exercise leads to a clear set of specific aims for future research.

Noninvasive assessment of sympathetic vasoconstriction in human and rodent skeletal muscle using near-infrared spectroscopy and Doppler ultrasound.

The precise role of the sympathetic nervous system in the regulation of skeletal muscle blood flow during exercise has been challenging to define in humans, partly because of the limited techniques available for measuring blood flow in active muscle. Recent studies using near-infrared (NIR) spectroscopy to measure changes in tissue oxygenation have provided an alternative method to evaluate vasomotor responses in exercising muscle, but this approach has not been fully validated. In this study, we tested the hypothesis that sympathetic activation would evoke parallel changes in tissue oxygenation and blood flow in resting and exercising muscle. We simultaneously measured tissue oxygenation with NIR spectroscopy and blood flow with Doppler ultrasound in skeletal muscle of conscious humans (n = 13) and anesthetized rats (n = 9). In resting forearm of humans, reflex activation of sympathetic nerves with the use of lower body negative pressure produced graded decreases in tissue oxygenation and blood flow that were highly correlated (r = 0.80, P < 0.0001). Similarly, in resting hindlimb of rats, electrical stimulation of sympathetic nerves produced graded decreases in tissue oxygenation and blood flow velocity that were highly correlated (r = 0.93, P < 0.0001). During rhythmic muscle contraction, the decreases in tissue oxygenation and blood flow evoked by sympathetic activation were significantly attenuated (P < 0.05 vs. rest) but remained highly correlated in both humans (r = 0.80, P < 0.006) and rats (r = 0.92, P < 0.0001). These data indicate that, during steady-state metabolic conditions, changes in tissue oxygenation can be used to reliably assess sympathetic vasoconstriction in both resting and exercising skeletal muscle.

Functional muscle ischemia in Duchenne and Becker muscular dystrophy.

Duchenne and Becker muscular dystrophy (DMD/BMD) comprise a spectrum of devastating X-linked muscle wasting disease for which there is no treatment. DMD/BMD is caused by mutations in the gene encoding dystrophin, a cytoskeletal protein that stabilizes the muscle membrane and also targets other proteins to the sarcolemma. Among these is the muscle-specific isoform of neuronal nitric oxide synthase (nNOSµ) which binds spectrin-like repeats within dystrophin's rod domain and the adaptor protein α-syntrophin. Dystrophin deficiency causes loss of sarcolemmal nNOSµ and reduces paracrine signaling of muscle-derived nitric oxide (NO) to the microvasculature, which renders the diseased muscle fibers susceptible to functional muscle ischemia during exercise. Repeated bouts of functional ischemia superimposed on muscle fibers already weakened by dystrophin deficiency result in use-dependent focal muscle injury. Genetic and pharmacologic strategies to boost nNOSµ-NO signaling in dystrophic muscle alleviate functional muscle ischemia and show promise as novel therapeutic interventions for the treatment of DMD/BMD.

Differential effects of nebivolol versus metoprolol on functional sympatholysis in hypertensive humans.

In young healthy humans, sympathetic vasoconstriction is markedly blunted during exercise to optimize blood flow to the metabolically active muscle. This phenomenon known as functional sympatholysis is impaired in hypertensive humans and rats by angiotensin II-dependent mechanisms, involving oxidative stress and inactivation of nitric oxide (NO). Nebivolol is a ß1-adrenergic receptor blocker that has NO-dependent vasodilatory and antioxidant properties. We therefore asked whether nebivolol would restore functional sympatholysis in hypertensive humans. In 21 subjects with stage 1 hypertension, we measured muscle oxygenation and forearm blood flow responses to reflex increases in sympathetic nerve activity evoked by lower body negative pressure at rest, and during rhythmic handgrip exercise at baseline, after 12 weeks of nebivolol (5-20 mg/d) or metoprolol (100-300 mg/d), using a double-blind crossover design. We found that nebivolol had no effect on lower body negative pressure-induced decreases in oxygenation and forearm blood flow in resting forearm (from -29±5% to -30±5% and from -29±3% to -29±3%, respectively; P=NS). However, nebivolol attenuated the lower body negative pressure-induced reduction in oxygenation and forearm blood flow in exercising forearm (from -14±4% to -1±5% and from -15±2% to -6±2%, respectively; both P<0.05). This effect of nebivolol on oxygenation and forearm blood flow in exercising forearm was not observed with metoprolol in the same subjects, despite a similar reduction in blood pressure. Nebivolol had no effect on sympathetic nerve activity at rest or during handgrip, suggesting a direct effect on vascular function. Thus, our data demonstrate that nebivolol restored functional sympatholysis in hypertensive humans by a mechanism that does not involve ß1-adrenergic receptors. Clinical Trial Registration- URL: http://www.clinicaltrials.gov. Unique identifier: NCT01502787.

Treatment with a nitric oxide-donating NSAID alleviates functional muscle ischemia in the mouse model of Duchenne muscular dystrophy.

In patients with Duchenne muscular dystrophy (DMD) and the standard mdx mouse model of DMD, dystrophin deficiency causes loss of neuronal nitric oxide synthase (nNOSµ) from the sarcolemma, producing functional ischemia when the muscles are exercised. We asked if functional muscle ischemia would be eliminated and normal blood flow regulation restored by treatment with an exogenous nitric oxide (NO)-donating drug. Beginning at 8 weeks of age, mdx mice were fed a standard diet supplemented with 1% soybean oil alone or in combination with a low (15 mg/kg) or high (45 mg/kg) dose of HCT 1026, a NO-donating nonsteroidal anti-inflammatory agent which has previously been shown to slow disease progression in the mdx model. After 1 month of treatment, vasoconstrictor responses to intra-arterial norepinephrine (NE) were compared in resting and contracting hindlimbs. In untreated mdx mice, the usual effect of muscle contraction to attenuate NE-mediated vasoconstriction was impaired, resulting in functional ischemia: NE evoked similar decreases in femoral blood flow velocity and femoral vascular conductance (FVC) in the contracting compared to resting hindlimbs (ΔFVC contraction/ΔFVC rest=0.88 ± 0.03). NE-induced functional ischemia was unaffected by low dose HCT 1026 (ΔFVC ratio=0.92 ± 0.04; P>0.05 vs untreated), but was alleviated by the high dose of the drug (ΔFVC ratio=0.22 ± 0.03; P<0.05 vs untreated or low dose). The beneficial effect of high dose HCT 1026 was maintained with treatment up to 3 months. The effect of the NO-donating drug HCT 1026 to normalize blood flow regulation in contracting mdx mouse hindlimb muscles suggests a putative novel treatment for DMD. Further translational research is warranted.

Tadalafil alleviates muscle ischemia in patients with Becker muscular dystrophy.

Becker muscular dystrophy (BMD) is a progressive X-linked muscle wasting disease for which there is no treatment. Like Duchenne muscular dystrophy (DMD), BMD is caused by mutations in the gene encoding dystrophin, a structural cytoskeletal protein that also targets other proteins to the muscle sarcolemma. Among these is neuronal nitric oxide synthase (nNOSµ), which requires certain spectrin-like repeats in dystrophin's rod domain and the adaptor protein α-syntrophin to be targeted to the sarcolemma. When healthy skeletal muscle is subjected to exercise, sarcolemmal nNOSµ-derived NO attenuates local α-adrenergic vasoconstriction, thereby optimizing perfusion of muscle. We found previously that this protective mechanism is defective-causing functional muscle ischemia-in dystrophin-deficient muscles of the mdx mouse (a model of DMD) and of children with DMD, in whom nNOSµ is mislocalized to the cytosol instead of the sarcolemma. We report that this protective mechanism also is defective in men with BMD in whom the most common dystrophin mutations disrupt sarcolemmal targeting of nNOSµ. In these men, the vasoconstrictor response, measured as a decrease in muscle oxygenation, to reflex sympathetic activation is not appropriately attenuated during exercise of the dystrophic muscles. In a randomized placebo-controlled crossover trial, we show that functional muscle ischemia is alleviated and normal blood flow regulation is fully restored in the muscles of men with BMD by boosting NO-cGMP (guanosine 3',5'-monophosphate) signaling with a single dose of the drug tadalafil, a phosphodiesterase 5A inhibitor. These results further support an essential role for sarcolemmal nNOSµ in the normal modulation of sympathetic vasoconstriction in exercising human skeletal muscle and implicate the NO-cGMP pathway as a putative new target for treating BMD.